Methods for inhibiting metastasis

ABSTRACT

The invention relates to methods for determining degree of integrin-mediated maintenance of cell invasive capacity of tumors, and to methods and compositions for inhibiting or preventing metastasis of cancers. In one aspect, the invention provides a method to determine degree of integrin-mediated maintenance of cell invasive capacity of epithelial derived tumors, particularly prostatic, breast, and lung cancers. In these regards, the invention relates to determining protein or mRNA of effectors of integrin-mediated maintenance of cell invasive capacity, particularly uteroglobin protein or mRNA, or to gauge degree of integrin-mediated maintenance of cell invasive capacity of prostatic, breast, or lung tumors. The invention also relates to methods and compositions, such as uteroglobin, which specifically bind to integrin and are effectors of integrin-mediated maintenance of tumor cell invasive capacity and thus inhibit metastasis.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/400,084, filed on Mar. 7, 1995 (now U.S. Pat. No. 5,696,092,issued on Dec. 9, 1997).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and compositions that providefor the treatment and inhibition of metastatic carcinoma. A particularaspect of the invention relates to methods and compositions containingcompounds which inhibit tumor cell proliferation and invasion,particularly those that are contain uteroglobin, uteroglobin muteins,uteroglobin mimetics, peptide analogs of uteroglobins and muteins.Further compositions of the invention include other types of activeingredients in combination with those described above.

2. Description of the Prior Art

Cancers develop from uncontrolled multiplication of cells. All cancersare life threatening. Even when cancer does not result in death, it ispermanently debilitating, not only to the patient, but also to family,friends and co-workers. Too often, moreover, cancers prove fatal. Thepersonal and public loss from this cluster of diseases, which cause asignificant fraction of all premature deaths, is beyond estimation.

Although effective treatment modalities have been developed in a fewcases, many cancers remain refractory to currently available therapies.Particularly difficult to treat are metastatic cancers. These cancerspose the highest risk to patients and, for optimal prognosis, often mustbe treated by aggressive methods that present increased risks ofdeleterious side-effects. Therefore, there is a great need for methodsthat accurately distinguish those tumors that are likely to metastasizefrom those that are unlikely to do so. Furthermore, methods for treatingmetastatic cancers often are inadequate, and there also is a clear needfor improved anti-metastatic agents and methods to treat metastaticcancers.

Metastatic cancers originate from a primary tumor. Metastasis of theprimary tumor produces secondary tumors and disseminated cancer. It iswell known that both primary and secondary tumors shed large numbers ofcells. The shed cells can spread through the body. For instance, aprimary tumor may damage the surrounding lymph or circulatory vessels,allowing entry of shed cells into the lymph or circulatory systems, andhastening their spread in the body. Moreover, shedding of cells bycancerous tumors increases during surgery and radiotherapy.

Most shed cells do not form new tumors. To do so such cells mustsurmount a series of physical and physiological barriers. In fact, aseries of distinct events must occur for metastasis to occur. Theprimary tumor physically must (i) invade interstitial space of theprimary tissue. In particular, it must (ii) penetrate the basementmembrane of the tissue. For most metastases the tumor must damage theendothelial cell wall of lymphatic or vascular vessels to provide accessto shed cells. Cells that enter the lymph or blood must (iii) survivehemodynamic stress and host defenses in the circulation and,furthermore, (iv) the cells must lodge at a new site in the circulatorysystem, a process that apparently involves aggregated platelets. A cellthen must (v) extravasate out of the vessel into the interstitial space.Finally, it must (vi) invade the interstitial space of the secondaryorgan and proliferate in the new location. Although the process ofmetastasis is physiologically complex, the overall pattern of metastasisis general to many types of cancers.

The metastatic process also clearly involves complex intracellularmechanisms that alter cancerous cells and their interactions withsurrounding cells and tissues. For instance, cancerous cells arecharacterized by aberrant expression of adhesion proteins, enzymes thatdegrade matrix components, autocrine factors, ligand-responsivereceptors, factors of angiogenesis and prostaglandins, to name a few. Inparticular, the signaling pathways that initiate tumor cell migrationare among the least understood aspects of invasion and metastasis.Currently, it is thought that proliferation of many cancerous cellsdepends upon specific ligand-receptor interactions. Thus far, however,it has not been possible to use this paradigm, or other concepts of theunderlying mechanisms of metastasis, to develop a therapy that preventsor effectively inhibits metastasis of metastatic cancers.

The complexity of the processes involved in metastasis, and the lack ofunderstanding of underlying molecular mechanisms, have made itparticularly difficult, in some cases, to distinguish tumors that arelikely to metastasize from those that are unlikely to do so. Theinability to discern the metastatic potential of tumors precludesaccurate prognosis and leads, inevitably, to the therapeuticintervention that either is too aggressive or insufficiently aggressive.Furthermore, for all types of cancers it has been difficult orimpossible, thus far, to develop treatments that inhibit or prevent thespread of metastatic tumors. Clearly, there remains a great need formethods to accurately determine the metastatic potential of tumors andfor effective anti-metastatic compositions and methods.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide methodsand compositions for inhibiting or treating metastasis.

Accordingly, the present invention relates to methods and compositionsthat provide for the treatment and inhibition of metastatic carcinoma. Aparticular aspect of the invention relates to methods and compositionscontaining compounds which inhibit tumor cell proliferation andinvasion, particularly those that are contain uteroglobin, uteroglobinmuteins, uteroglobin mimetics, peptide analogs of uteroglobins andmuteins. Further compositions of the invention include other types ofactive ingredients in combination with those described above.

The present invention also relates to methods for gauging the metastaticpotential of tumors of epithelial cell origin by determining an effectorof integrin-mediated maintenance of cell invasive capacity of atumor-containing tissue. This aspect of the invention particularlyrelates to determining uteroglobin protein or mRNA in cells of a biopsysample to determine metastatic potential of a prostatic, breast, or lungtumor.

The present invention further relates to methods and compositions thattreat or inhibit metastases of cancers of epithelial cell origin,especially human cancers of the prostate, breast and lung. A particularaspect of the invention relates to methods and compositions thatfacilitate or restore control levels of integrin-mediated maintenance ofcell invasive capacity. In one aspect in this regard, the inventionparticularly relates to methods and compositions that replenish controllevels of non-aberrant uroglobin that are diminished in patients havingmetastatic cancers of epithelial origin. Compositions of the inventionalso particularly include those that contain uteroglobin, uteroglobinmuteins, and peptide analogs of uteroglobins that restore control levelsof non-aberrant uteroglobin in metastatic cancer patients. Furtheruseful in this regard are compositions that contain mimetics ofuteroglobin, particularly of human uteroglobin. Further compositions ofthe invention include other types of active ingredients in combinationwith those that restore normal levels of non-aberrant uteroglobin.

The invention also particularly relates to methods to treat or inhibitmetastases of human cancers of epithelial cell origin by administeringthe foregoing compositions. Especially in this regard the inventionrelates to methods using human uteroglobin to inhibit or preventmetastasis of human prostate, breast and lung cancers. Further, thisaspect of the invention may be accomplished by genetic therapy.

Methods and compositions of the invention may be used by themselves andwith other treatment modalities.

In accomplishing the foregoing object, there has also been provided, inaccordance with one aspect of the present invention, a method foridentifying an effector of integrin-mediated tumor maintenance of cellinvasive capacity, comprising the step of administering to an organismsuffering from a cancer of epithelial cell origin a compound that bindsbinds to integrin and in an amount and manner effective to identify thedegree of integrin-mediated maintenance of tumor cell invasive capacity.

Another method of the present invention is directed to a diagnostic kitfor the detection of an effector of integrin-mediated tumor cellinvasive capacity in a biopsy sample, the kit comprising: a firstreagent that binds specifically to an effector of integrin-mediatedmaintenance of cell invasive capacity in a biopsy sample prepared fordetermination of the effector, and a second reagent for detectablylabeling the primary binding reagent bound specifically to cells in thebiopsy sample, wherein the determination of the effector is diagnosticof integrin-mediated maintenance of cell invasive capacity.

In certain preferred embodiments of the kits of the invention, theeffector is a molecule which binds to integrin, among which uteroglobinis particularly preferred.

In certain further preferred embodiments of the this aspect of theinvention, the first reagent is an antibody. In these embodiments, thedetermination would occur where uteroglobin-antibody staining wouldindicate normal integrin-mediated maintenance of cell invasive capacityof an non-aggressive cancer if strong staining occurred, and aberrantintegrin-mediated maintenance of cell invasive capacity of an aggressivecancer if a weak or no signal occurred.

Additional preferred embodiments of this aspect of the invention arethose in which the first reagent is a hybridization probe. In certainpreferred embodiments of this aspect of the invention, the effector is amolecule which binds to integrin, among which uteroglobin isparticularly preferred.

Another preferred embodiment of the present invention is directed to amethod for treating or inhibiting metastasis of a cancer of epithelialcell origin, comprising the step of administering to an organismsuffering from a cancer of epithelial cell origin a compound thatrestore/replenish control levels of non-aberrant uroglobin in an amounteffective to inhibit or treat metastasis of the tumor.

In a preferred embodiment of an aspect of the invention in this regard,the compound binds to integrin. In especially highly preferredembodiments of this aspect of the invention, the compound is auteroglobin, a mutein of a uteroglobin, a peptide analog of auteroglobin, or a mimetic of uteroglobin. Uteroglobin is preferred andhuman uteroglobin is particularly highly preferred in this regard.

In another regard, preferred embodiments of the present method are thoseused to treat a cancers of the prostate, breast, and lung in a humanpatient.

In further preferred embodiments, the method is used in conjunction withanother treatment. In this regard, preferred treatments include surgicalintervention, radiation therapy, hormonal therapy, immunotherapy,chemotherapy, cryotherapy or gene therapy.

In accordance with another aspect of the present invention, there hasbeen provided a pharmaceutical composition for inhibiting or preventingmetastasis of a cancer of epithelial cell origin, comprising: (i) acompound that binds to integrin on cells of a tumor of epithelial cellorigin effective to inhibit or treat metastasis of the tumor in anorganism and (ii) a carrier for effective the therapeutic administrationof the compound to the organism.

In certain preferred embodiments of the invention the compound binds tointegrin. In this regard, the preferred embodiment of the compound is auteroglobin, a mutein of a uteroglobin, a peptide analog of auteroglobin, or a mimetic of uteroglobin. Among these, uteroglobins arevery highly preferred, and human uteroglobins are among the most highlypreferred compounds of the present invention.

In accordance with another aspect of the invention there has beenprovided a method for determining the degree of integrin-mediatedproliferation of tumors, particularly those of epithelial cell origin.In certain preferred embodiments of this aspect of the invention therehas been provided a method for determining the degree ofintegrin-mediated proliferation of tumors of epithelial cell origincomprising the steps of (A) determining an effector of integrin-mediatedmaintenance of cell invasive capacity in a biopsy sample of a tumor; (B)comparing effector in tumor cells in the biopsy sample with effector incontrol cells, and (C) determining degree of integrin-mediatedmaintenance of cell invasive capacity, wherein effector in the tumorcells characteristic of control cells of non-aggressive tumors indicateshigh degree of integrin-mediated maintenance of cell invasive capacityand effector in the tumor cells characteristic of control cells ofaggressive indicates low degree of integrin-mediated maintenance of cellinvasive capacity.

In some preferred embodiments of this aspect of the invention theeffector binds to integrin. In particularly preferred embodiments inthis regard, the effector is uteroglobin.

In certain preferred embodiments the effector is determined by assayingthe effector protein in cells of the tumor. In particularly preferredembodiments in this regard, the effector binds to integrin. Especiallypreferred is uteroglobin. In particularly preferred embodiments in thisregard the tumor is a prostatic, breast, or lung tumor and the effectoris uteroglobin.

In another aspect of the invention, preferred embodiments of theinvention provide methods for determining degree of integrin-mediatedmaintenance of cell invasive capacity in which a protein is assayed byimmunocytochemistry. In certain preferred embodiments of this type, theeffector binds to integrin. Particularly preferred in embodiments of theinvention in this regard is uteroglobin. In particularly preferredembodiments in this regard the tumor is a prostatic, breast, or lungtumor and the effector is uteroglobin.

In certain additional preferred embodiments of the invention in thisregard, the effector is determined by assaying an mRNA in cells of atumor. In particularly preferred embodiments in this regard, the mRNAencodes a molecule that binds to integrin. Especially preferred isuteroglobin. In particularly preferred embodiments in this regard thetumor is a prostatic, breast, or lung tumor and the effector isuteroglobin.

In certain preferred embodiments in this regard, the mRNA is determinedby a method comprising a step of hybridizing a probe to cells fixed on asurface. In certain preferred embodiments of this aspect of theinvention the mRNA is determined by in situ hybridization.

In preferred embodiments of the invention in both regards the effectorbinds to integrin, most particularly uteroglobin. In particularlypreferred embodiments in this regard the tumor is a prostatic, breast,or lung tumor and the effector is uteroglobin.

In another aspect of the invention in this regard, aberrant mRNA isdetermined. In preferred embodiments of the invention in this regard,the mRNA encodes binds to integrin, most particularly uteroglobin. Inparticularly preferred embodiments in this regard the tumor is aprostatic, breast, or lung tumor and the effector is uteroglobin.

In a still further object of the invention there has been provided a kitfor determining degree of integrin-mediated maintenance of cell invasivecapacity of a tumor. In certain preferred embodiments kits of theinvention comprise: (A) a first reagent that binds specifically to aneffector of integrin-mediated maintenance of cell invasive capacity in abiopsy sample prepared for determination of the effector, and (B) asecond reagent for detectably labelling the primary binding reagentbound specifically to cells in the biopsy sample, wherein thedetermination of the effector tumor is diagnostic of the degree ofintegrin-mediated maintenance of cell invasive capacity of the tumor.

In certain preferred embodiments of the kits of the invention, theeffector binds to integrin, among which uteroglobin is particularlypreferred.

In certain further preferred embodiments of the this aspect of theinvention, the first reagent is an antibody. In particularly preferredembodiments in this respect, the effector binds to integrin, among whichuteroglobin is particularly preferred.

Additional preferred embodiments of this aspect of the invention arethose in which the first reagent is a hybridization probe. In certainpreferred embodiments of this aspect of the invention, the effectorbinds to integrin, among which uteroglobin is particularly preferred.Other objects, features and advantages of the invention will be apparentfrom the following detailed description. It should be understood,however, that the detailed description and the specific examples, whileproviding general and specific descriptions and indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of theinvention will become apparent to those skilled in the art from thedetailed description and other aspects of the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar chart showing the dose effect of uteroglobin on theinvasiveness of cells of the TSU-Pr1 and DU-145 cell lines, epithelialcell lines derived from human prostate tumors. Both TSU-Pr1 and DU-145cells are androgen independent. Cells were cultured in zero, 0.01, 0.1and 1.0 μM uteroglobin for 24 hr. Invasiveness then was assayed bymigration through filters coated with reconstituted basement membrane(RBM) in response to serum free or fibroblast conditioned media (FCM).Invading cells were stained with crystal violet, the dye was extractedand invasiveness was determined by measuring dye concentration byoptical absorbance at 585 nm. Each point in the graph is the mean ofresults of three separate experiments, each carried out using triplicatecultures. The bars show the standard error of the mean for each point.

FIG. 2 is a bar chart showing the dose effect of uteroglobin on theinvasiveness of cells of the PC3-M and LNCaP cell lines, epithelial celllines derived from human prostate tumors. LNCaP cells areandrogen-sensitive. PC3-M cells are androgen independent. Assays wereperformed as described in the caption to FIG. 1.

FIG. 3 is a bar chart showing that myoglobin, albumin andheat-inactivated uteroglobin do not affect the invasiveness of DU-145cells. Assays were performed essentially as described in the caption toFIG. 1.

FIG. 4 is a graph showing a time course of the effect of uteroglobin oninvasiveness of FCM-stimulated DU-145 cells. Cells were incubatedwithout or with 1.0 μM uteroglobin for 3, 6, 12, or 24 hr. and thenassayed for invasion in response to FCM as described in the caption toFIG. 1.

FIG. 5 is a bar graph showing that uteroglobin is bound to integrin invivo. FIG. 5 unexpectedly shows that a positive uteroglobin signal wasobtained in samples of normal tissue (100%) but which was remarkablydiminished in tumor tissue (20%). FIG. 5 shows that uteroglobin is boundto integrin in vivo since uteroglobin co-immunoprecipitated withanti-integrin antibody.

GLOSSARY

The abbreviations and terms in the present disclosure are employed incontemplation of their fullest meaning consistent with the disclosed andclaimed invention. The following brief explanations are entirelyillustrative and neither exhaustively define nor limit the inventiondisclosed and claimed herein. The full meaning of the terms will beclear from an understanding of the invention based on contemplation ofthe disclosure as a whole in light of a full understanding of thepertinent arts.

DU-145: an epithelial cell line derived from a human prostate tumor,which is androgen independent.

LNCaP: an epithelial cell line derived from a human prostate tumor,which is androgen sensitive. The LNCaP cell line was derived from asupraclavicular lymph node metastasis of a human prostate carcinoma.Cells of this line exhibit increased proliferation in response toandrogen, in vitro, and they secrete prostate specific antigen (PSA), amarker of differentiated epithelial cells.

TSU-Pr1: an epithelial cell line derived from a human prostate tumor,which is androgen independent.

PC3-M: an epithelial cell line derived from a human prostate tumor,which is androgen independent.

EFFECTOR: a substance that engenders, alters, modulates or controls anactivity of a cell; a substance that can engender, alter, modulate orcontrol a physiological activity of a cell or organism. Typically, aprotein, such as an enzyme, cofactor or transcription regulatoryprotein, or an activator or inhibitor of an enzyme, an enzyme complex, areceptor or a receptor complex, for instance.

EPITHELIAL CELL ORIGIN: derived from an epithelial cell, of whatevertissue.

EXTRACELLULAR MATRIX (ECM): a collective organization of membrane-boundand membrane-associated molecules which are found at the outer surfaceof most cells. ECM molecules include lipids, sacharrides, aminoacids,proteins, lipoproteins, glycolipids, glycoproteins, andglycolipoproteins. Specific examples of ECM molecules include integrins,cadherins, selecting, immunoglobulins, collagens, fibronectin, andlaminin. The ECM mediates a wide range of cellular processes includingtransmembrane signal transduction, cell adhesion, migration, spreading,differentiation, proliferation, and tumor metastasis. The primarymediator of these ECM-regulated cellular processes are membrane-boundintegrin molecules.

FCM: fibroblast conditioned media

Control: a reference against which a test outcome is compared to gaugeresults. A control series is a plurality of such references thatrepresent points along a qualitative or a quantitative scale.

IDENTIFYING: in the context of identifying PIN, ascertaining,establishing or otherwise determining one or more factualcharacteristics of prostatic intraepithelial neoplasia or a similarintermediate condition.

INHIBITION: inhibition of metastasis may be measured by many parametersin accordance with the present invention and, for instance, may beassessed by delayed appearance of secondary tumors, slowed developmentof primary or secondary tumors, decreased occurrence of secondarytumors, slowed or decreased severity of secondary effects of disease,arrested tumor growth and regression of tumors, among others. In theextreme, complete inhibition, is referred to herein as prevention.

INTEGRIN: Integrins are a family of noncovalently associatedheterodimers of α and β peptide chains. Integrins are a family ofadhesion receptors known to mediate a variety of cell-cell interactionsand cell interactions with extracellular matrix (ECM) ligand proteins,such as laminin, fibronectin, collagen, and fibrinogen (Brodt, P. andDedhar, S., Chapter 4, The Integrins: Mediators of Cell-ExtracellularMatrix and Intercellular Communication, pp35-60 in Cell Adhesion andInvasion in Cancer Metastasis, Brodt, P., Ed., R. G. Landes Company,Austin, Tex., 1996). Integrins bind to the RGD (Arg-Gly-Asp) sequence ofthe ligand protein. Integrins have been implicated in several differentmodes of signal transduction across the ECM. In addition, integrins havebeen shown to be involved in several adhesion cascades involving one ormore other types of adhesion receptors. Integrin maintains extracellularmatrix (ECM) integrity. Research findings indicate that multistepintegrin-mediated interactions between cancer cells, vascularendothelium and target organ parenchyma may contribute to thedissemination, proliferation, and invasive capacity of cancer cellsduring: (1) detachment of individual cells from a primary tumor; (2)invasion of local tissue or stroma; (3) entry of tumor cells intovascular blood vessels or lymphatic channels; (4) arrest and adhesion tothe vasculature and extravasation out of the vasculature; and (5)invasion through basement membrane and into parenchyma of the targetorgan. In particular, evidence suggests that integrin mediates themaintenance of tumor cell invasive capacity. Inhibition of integrins maybe useful in preventing tumor invasiveness by limiting the ability oftumor cells to adhere to vascular endothelium and thereby inhibitingextravasation, invasion and metastasis.

METASTASIS: As set out in Hill, R. P., Chapter 11, Metastasis, pp178-195in The Basic Science of Oncology, Tannock et al., Eds., McGraw-Hill, NewYork (1992), which is incorporated by reference herein in its entirety,metastasis is “The ability of cells of a cancer to disseminate and formnew foci of growth at noncontiguous sites (i.e., to form metastases).”

Similarly, metastasis is described in Aznavoorian et al., Cancer 71:1368-1383 (1993), which is incorporated by reference herein in itsentirety, as “The transition from in situ tumor growth to metastaticdisease is defined by the ability of tumor cells of the primary site toinvade local tissues and to cross tissue barriers. . . . To initiate themetastatic process, carcinoma cells must first penetrate the epithelialbasement membrane and then invade the interstitial stroma. . . . Fordistant metastases, intravasation requires tumor cell invasion of thesubendothelial basement membrane that must also be negotiated duringtumor cell extravasation . . . . The development of malignancy is alsoassociated with tumor-induced angiogenesis [which] not only allows forexpansion of the primary tumor, but also permits easy access to thevascular compartment due to defects in the basement membranes of newlyformed vessels.”

MIMETIC: a molecule which, in shape and effect, mimics the shape andtherefore the activity of another molecule or complex of molecules uponwhich it is designed.

MUTEIN: An amino acid sequence variant of a protein. The variation inprimary structure may include deletions, additions and substitutions.The substitutions may be conservative or non-conservative. Thedifferences between the natural protein and the mutein generallyconserve desired properties, mitigate or eliminate undesired propertiesand add desired or new properties. In the present invention the muteinsgenerally are those that maintain or increase anti-metastatic activity.Particularly, uteroglobin muteins are amino acid sequence variants ofuteroglobin that maintain or increase the anti-metastatic activity ofuteroglobin.

PEPTIDE ANALOG: an oligo or polypeptide having an amino acid sequence ofor related to a protein. Peptide analogs of the present invention arepeptides that have anti-metastatic activity and an amino acid sequencethe same as or similar to a region of an anti-metastatic protein, suchas uteroglobin.

PREVENTION: in relation to metastasis, virtually complete inhibition, nometastasis if it had not occurred, no further metastasis if there hadalready been metastasis of a cancer. See INHIBITION.

PSA: Prostate specific antigen

RBM: reconstituted basement membrane. A multicomponent matrixapproximating the molecular composition of the intracellular tissuematrix and the epithelial cell basement membrane. Preparations forpreparing RBM are well known and are available from commercialsuppliers.

SFM: Serum free medium

UG: uteroglobin

hUG: human uteroglobin

DETAILED DESCRIPTION OF THE INVENTION

Notwithstanding past failures to develop methods to distinguishnon-metastatic aberrations and tumors with high degree ofintegrin-mediated maintenance of cell invasive capacity from aberrationsand tumors with low degree of integrin-mediated maintenance of cellinvasive capacity, the present invention provides methods fordetermining the degree of integrin-mediated maintenance of cell invasivecapacity of aberrant growths, tumors and cancers.

In addition, notwithstanding past failures to develop effectiveanti-metastatic treatments, the present invention provides compounds andcompositions for inhibiting cancer metastases and methods foradministering the compounds and compositions to inhibit or preventmetastasis of a tumor of epithelial cell origin in an organism.

DETERMINING THE DEGREE OF INTEGRIN-MEDIATED

Maintenance of cell invasive capacity OF TUMORS

Determining the activity of factors that effect integrin-mediatedmaintenance of cell invasive capacity can serve to indicate degree ofintegrin-mediated maintenance of cell invasive capacity of a tumor. Inthis regard, determining the amount of effector bound to integrin, andthe activity or abundance of factors that influence the effector boundto integrin can serve to indicate degree of integrin-mediatedmaintenance of cell invasive capacity. Determining degree ofintegrin-mediated maintenance of cell invasive capacity in accordancewith this aspect of the present invention is illustrated by thefollowing discussion of uteroglobin protein, mRNA or DNA as an index ofdegree of integrin-mediated maintenance of cell invasive capacity ofprostatic, breast, or lung tumors, a very particularly preferredembodiment of the invention, which should not be construed as beinglimitative.

METHODS

Uteroglobin to index decree of integrin-mediated maintenance of cellinvasive capacity

The prostate is the sex gland in males that makes seminal fluid. It islocated, in the standing male, vertically below the bladder, where itsurrounds the urethra. Generally, it is shaped roughly like a slightlyelongated sphere, like a walnut. In most men, it is about an inch indiameter until about age 50 and thereafter it tends to grow larger.

Pathology of the prostate can present as infection, benign enlargementor cancer. Benign growth adversely affects health only when the enlargedprostate constricts the urethra and interferes with urination. Malignantgrowth always poses a threat to health and life of the patient;although, as discussed below, prognoses and indicated treatments varygreatly between occurrences.

Prostate cancer is the most frequently diagnosed cancer in men in theUnited States. Prostate cancers generally do not grow quickly. Usually,they double in size only every three or four years. Adverse affects ofprostate cancer also develop slowly, as they are effected by the growthof the tumor itself.

The slow progression of prostate cancer presents something of aconundrum in men 50 or older, who present the majority of prostatecancer cases. Often the normal progression of prostate cancer suggeststhat debilitating effects will not develop within the normal lifeexpectancy of the patient. Given the lack of effective treatments andthe deleterious side effects attendant to the treatments currentlyavailable, waiting may be the best therapy for many elderly patients.

Unfortunately, it is difficult to distinguish benign from canceroustumors and, more importantly, slow growing localized tumors from thoseformed by more aggressive, metastatic cancers. Thus, even the bestphysician cannot accurately predict the course of progression of a givenprostate cancer, and cannot prescribe the best treatment regimen.

In one aspect the present invention overcomes this obstacle to effectivetreatment of such tumors by providing a method to determine the degreeof integrin-mediated maintenance of cell invasive capacity of prostatic,breast, or lung tumors. In accordance with this aspect of the invention,uteroglobin protein, mRNA or DNA is determined in cells of biopsymaterial. The protein, mRNA or DNA determined in the cells, bycomparison to uteroglobin determined in normal cells, indicates thedegree of integrin-mediated maintenance of cell invasive capacity ofprostatic, breast, or lung tumors, particularly those of epithelial cellorigin.

It is worth noting in this respect that previous studies did notidentify the relationship between degree of integrin-mediatedmaintenance of cell invasive capacity of a tumor and decreasedexpression of uteroglobin (or any other effectors of integrin-mediatedmaintenance of cell invasive capacity). In previous studies, forinstance, uteroglobin (called Clara cell 10 kDa protein, abbreviatedCC10) was used as a marker for certain types of cells, and cell-typespecificity of its expression was studied. (As described in Linnoila etal., A.J.C.P. 97(2): 235-243 (1992) and Peri et al., J. Clin. Invest.92: 2099-2109 (1993), which are incorporated by reference herein intheir entirety). In addition, CC10 expression was reported to varybetween patients and cell types. In particular, it was reported thatCC10 expression was lower in lung cancer patients and in smokers withoutlung cancer than it was in non-smokers, and decreased CC10 expressionhas been loosely associated with neoplasm. (Broers et al., Lab. Invest.66: 337-346 (1992) and Jensen et al., Int. J. Cancer 58: 629-637 (1994),which are incorporated by reference herein in their entirety. However,no studies of CC10 expression have suggested that uteroglobin expressionin cells of a tumor can be used to determine degree of integrin-mediatedmaintenance of cell invasive capacity.

Specific detection of proteins

Proteins indicative of degree of integrin-mediated maintenance of cellinvasive capacity of tumors can be determined in cells in biopsymaterial by conventional methods well known to those of skill in theart. Such methods are described in many standard textbooks andlaboratory manuals. For instance, the techniques for making and usingantibody and other immunological reagents and for detecting particularproteins in samples using such reagents are described in CURRENTPROTOCOLS IN IMMUNOLOGY, Coligan et al., Eds., John Wiley & Sons, NewYork (1995), which is incorporated by reference herein in partspertinent to making and using reagents useful for determining specificproteins in samples. As another example, immunohistochemical methods fordetermining proteins in cells in tissues are described in Volume 2,Chapter 14 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al.,Eds., John Wiley & Sons, Inc. (1994), which is incorporated by referenceherein in part pertinent to carrying out such determinations. Finally,Linnoila et al., A.J.C.P. 97 (2): 235-243 (1992) and Peri et al., J.Clin. Invest. 92: 2099-2109 (1993), incorporated herein as referred toabove, describe techniques that may used, in part, in this aspect of thepresent invention.

For instance, uteroglobin can be determined in sample in accordance withthe invention by histochemical methods set out in Miyamoto et al., J.Urology 149: 1015-1019 (1993), which is incorporated by reference hereinin its entirety. As described therein, for instance, suitable biopsymaterial is obtained from a patient suspected of having benign prostatichyperplasia or prostatic carcinoma and immediately placed into 0.01Mphosphate buffered saline. Thereafter, the material is immediatelyprocessed. It is mounted on a brass plate using rat liver homogenate asan adhesive. The material then is frozen in liquid nitrogen-cooledisopentane. Sections suitable for assay of uteroglobin in cells of thematerial are sectioned in a cryostat. Sections are obtained across thebiopsy material, avoiding parts of the biopsy material that are damagedor deleteriously altered by the removal process.

Sections are dried at room temperature, fixed and then washed.Paraformaldehyde is a particularly useful fixative in this regard, butmany other fixatives also can be used. The sections may be pretreatedwith hydrogen peroxide and a non-ionic detergent, such as Triton X-100.Also, sections may be incubated with a blocking solution to reducenon-specific binding. For instance, the sections may be incubated withgoat blocking serum prior to incubation with a goat serum, goat antibodyor goat antibody-derived reagent.

Uteroglobin then is visualized for determination in the samples using auteroglobin-specific binding reagent, such as a monoclonal or apolyclonal anti-uteroglobin antibody. Binding of theuteroglobin-specific reagent to cells in the sections may be determineddirectly, if the reagent has been conjugated to a detectable label, orusing a second or additional reagents, such as a secondaryantibody-enzyme conjugate.

In preferred embodiments of the invention, the uteroglobin-specificreagent is an antiserum, a polyclonal antibody, a derivative of apolyclonal antibody, a monoclonal antibody, a derivative of a monoclonalantibody or an engineered antibody, such as a single chain antibody.Derivatives of monoclonal and polyclonal antibodies include conjugatesand fragments. Antibodies conjugated to detectable labels are preferredin this regard. Among detectable labels are enzymes such as horseradishperoxidase. Among fragments preferred in this regard are Fab fragments,F(ab′)₂ fragments and F(ab′) fragments.

Sections are incubated with uteroglobin-specific reagent underconditions effective for the uteroglobin-specific reagent to bindefficiently to uteroglobin in said cells, while binding to othercellular components is inefficient; i.e., under conditions effective forthe ratio of specific to non-specific binding to provide accuratedetermination of uteroglobin content in cells of the biopsy material.

At the same time, control sections may be incubated under the sameconditions with a corresponding reagent that is not specific foruteroglobin, to estimate background binding. For polyclonal immuneserum, for instance, control sections can be incubated with preimmuneserum to monitor background, non-specific binding. After the incubationperiod, the specific reagent, and any reagent used in the controls, isremoved, as by washing.

If the primary, uteroglobin-specific reagent is detectably labelled,then the label may be determined and, thereby the uteroglobin content ofcells in the sample. In this case, controls preferably would be labeledand would be determined in like fashion. More often, and preferably, asecondary reagent is used to visualize binding of reagents on thesections, as described below.

After removing unbound specific and non-specific reagents, test andcontrol sections are incubated with a secondary reagent that bindsspecifically to the primary, uteroglobin specific reagent and itscounterpart in the controls. Preferably, the secondary reagent is abiotinylated anti-antibody.

The sections are incubated with the secondary reagent under conditionsfor the reagent to bind efficiently to the primary reagent (and itscounterpart in the controls) in the cells, while binding to othercellular components is inefficient; i.e., under conditions effective forthe ratio of specific to non-specific binding to provide accuratedetermination of uteroglobin content in cells of the biopsy material.

Thereafter, the unbound fraction of the secondary reagent is removedfrom the sections. The secondary reagent, and its counterpart in thecontrols, then is determined. If the secondary reagent comprises adetectable label, incubation with a tertiary reagent generally will notbe necessary. However, use of a tertiary reagent comprising a detectablelabel is more commonly employed for immunocytochemical analysis,generally. Therefore, for illustrative purposes, the three componentassay is described here.

The sections then are incubated with a tertiary reagent comprising adetectable label that binds specifically to the secondary reagent.Incubation is carried out under conditions effective for the tertiaryreagent to bind efficiently to the secondary reagent bound to primaryreagent in cells in the test sections or its counterpart in controlsections, while binding to other cellular components is inefficient;i.e., under conditions effective for the ratio of specific tonon-specific binding to provide accurate determination of uteroglobincontent in cells of the biopsy material. A preferred tertiary reagentcomprising a detectable label is an avidinated enzyme for binding tobiotinylated secondary reagent. A preferred enzyme in this regard ishorseradish peroxidase.

Unbound tertiary reagent is removed, by washing the sections withbuffer, for instance. The detectable label bound in cells in the biopsymaterial then may be determined. In preferred embodiments of theinvention, sections are incubated under conditions effective for anenzyme in the tertiary reagent to catalyze a chromogenic reaction.Binding of the uteroglobin-specific reagent is determined by the colorgenerated by the reaction.

Suitable reagents and conditions for carrying out the determination ofuteroglobin in cells in biopsy samples are well known and readilyavailable. A multiplicity of procedures and reagents can be effectivelyemployed for this purpose. Such reagents and techniques routinely areemployed by those of skill in the arts of immunocytochemistry,histopathology and cytology.

Kits for performing such assays, in whole and in part, are widelyavailable from numerous commercial suppliers. Incubation with secondaryantibodies, and subsequent visualization of uteroglobin, can be carriedout according the given procedures prescribed by commercial suppliers.

In preferred embodiments the sections are stained with hematoxylin andeosin to confirm pathology and to facilitate comparison of uteroglobinin normal and diseased cells in the same section.

In particularly, preferred embodiments, the relative staining ofdiseased and normal cells in a sections is compared with staining incontrol cells. The control cells are reference standards which typifyresults obtained by a given procedure in normal cells, cellscharacteristic of non-aggressive malignant tumors, and cellscharacteristic of aggressive malignant tumors. Within any category,moreover, control cells may provide a graded series of characteristicresults. Uteroglobin in control cells may be determined at the same timeuteroglobin is determined in cells of the biopsy sample, or at anothertime. In a particularly preferred embodiment of the invention,uteroglobin is determined in control cells which serve as a standardreference series for subsequent clinical assays.

In normal tissue immunocytochemical techniques, such as those describedabove, reveal very heavy staining of uteroglobin in the luminal surfaceof prostatic, breast, or lung epithelial cells. Biopsy material frommalignant tumors shows significant decreases in staining of uteroglobinin cells. The decrease in staining of the luminal surface of epithelialcells in prostatic tumors is particularly dramatic. Compared tonon-aggressive malignant tumors, uteroglobin staining either cannot bedetected or is more faint in the same cells in aggressive malignantprostatic tissue.

The risk of developing invasive cancer is gauged by the decrease inuteroglobin in diseased cells in the biopsy sample relative to cells innormal tissue of the same type, as described above. In biopsy materialcontaining both normal and diseased tissue the staining of cells in thenormal and diseased tissue can be compared on the same section. Ingeneral, the cells in low grade relatively confined tumors expressuteroglobin in amounts similar to normal cells. The cells in aggressive,invasive tumors express little or no uteroglobin and are poorlydifferentiated in their morphology.

Specific detection of mRNA and DNA

mRNA also can be determined in cells in biopsy samples to determinedegree of integrin-mediated maintenance of cell invasive capacity. mRNAcan be determined by a variety of methods well known to those of skillin the art, which can be carried out using well known and readilyavailable starting materials, including those widely available fromcommercial suppliers. Techniques useful in this regard are described inthe foregoing references. Techniques that may be particularly pertinentin this regard relating to uteroglobin are described in Broers et al. ,Lab. Invest. 66: 337-346 (1992) and Jensen et al., Int. J. Cancer 58:629-637 (1994), incorporated herein as referred to above.

A given mRNA may determined in cells of biopsy tissue by in situhybridization to a specific probe. Such probes may be cloned DNAs orfragments thereof, RNA, typically made by in vitro transcription, oroligonucleotide probes, usually made by solid phase synthesis. Methodsfor making and using probe suitable for specific hybridization in situare ubiquitously known and used in the art.

By specific hybridization is meant that the probe forms a duplex withthe given, target mRNA that is stable to the conditions of hybridizationand subsequent incubations and that duplexes formed between the probeand other, non-target mRNAs are not stable and generally do not persistthrough subsequent incubations. Specific hybridization thus means thatthe ratio of hybridization to target and non-target mRNAs provides anaccurate determination of the target mRNA in cells in the biopsy sample.

In a particularly preferred embodiment of the present invention a probethat hybridizes specifically to uteroglobin mRNA is used to determineuteroglobin mRNA in cells of biopsy tissue, particularly prostatic,breast, or lung biopsy material.

Techniques suitable for in situ determination of target mRNAs, such asuteroglobin mRNA, are described in a variety of well known and readilyavailable laboratory manuals, as well as the primary literature. Anillustrative procedure from the primary literature in this regard isdescribed in Broers et al. Laboratory Investigation 66 (3): 337-346(1992), which is incorporated by reference herein in its entirety.

In general, biopsy material is obtained by suitable surgical procedureand snap frozen, as by freezing in methybutane/dry ice. The samples canbe embedded and sectioned much as described above for the determinationof protein is biopsy samples. Sections can be thawed onto and affixed toglass slides previously cleaned with acid and ethanol and coated withpoly-L-lysine. The tissue sections thereafter can be exposed to bufferedformaldehyde, acetylated, treated with buffered glycine and thenprehybridized in 50% formamide, 2×SSC (where 1×SSC is 0.15M NaCl, 0.015Msodium citrate, pH 7.0). After prehybridization the sections can behybridized to the labelled probe in 50% formamide, 10% dextran sulfate,2×SSC.

The exact conditions of the steps in the procedure, especially theprehybridization, hybridization and criterion steps will be adjustedwith the T_(m) (or the T_(d)) of the probe and to provide the desireddegree of specificity of hybridization; i.e., the desired stringency.

Theoretical approximations and empirical methods for determining properconditions in this regard are well known and routinely practiced bythose skilled in the pertinent arts. Approximation calculations andexperimental techniques in this regard are described, for instance, inSambrook et al. (1989) referred to herein above.

Those of skill will appreciate, for instance, that the formamide in theforegoing solutions serves to provide equivalent hybridizationconditions at lower temperature. For instance, hybridization in 50%formamide at about 50° C. provides conditions similar to hybridizationat 65° C. without formamide. The lower temperature of hybridization canhelp preserve the biopsy sections during the hybridization procedure,aiding subsequent identification and examination of cells and mRNAcontent. Other agents that preserve features of the tissue sections thataid analysis likewise are preferred.

Dextran sulfate generally is used to accelerate the hybridizationreaction and to drive it to completion in a shorter period of time, asis well known. Similar agents that increase the rate of hybridization,consistent with accurate determination of specific mRNA content, alsoare useful in the present invention.

Following hybridization, the probe-containing solution and unbound probeare removed. Typically, the sections are washed several times withprehybridization buffer, such as 50% formamide, 2×SSC, at or slightlyabove the hybridization temperature.

If an RNA probe is used for detection of the target mRNA, the sectionsthen are treated with RNAseA, typically in the same solution, and thenwashed to remove RNAseA and byproducts with 50% formamide, 2×SSC underthe same conditions as the previous washings.

Finally, the sections typically are washed several additional times in2×SSC at room temperature and then air dried.

Radioactive probes. generally are visualized by autoradiography. Forthis purpose slides can be dipped in a photographic emulsion, dried andallowed to expose the emulsion at 4° C. for an appropriate period oftime. Using a preferred emulsion, NTB-2 nuclear track emulsion, exposuretimes of 3 to 7 days are appropriate. The exposure time can be alteredby a variety of factors including the use of more highly labelledprobes.

The emulsions are developed at the end of the exposure period and then,typically, counterstained with hematoxylin and eosin. Subsequently,labelling of target mRNA in cells can be assessed by microscopy usingbrightfield and darkfield illumination.

As discussed above regarding protein, the abundance and distribution ofthe target mRNA in cells in the biopsy section indicates the degree ofintegrin-mediated maintenance of cell invasive capacity of the tumor.Particularly, the relative abundance of mRNA in diseased and normalcells indicates degree of integrin-mediated maintenance of cell invasivecapacity.

A variety of controls may be usefully employed to improve accuracy inassays of this type. For instance, sections may be hybridized to anirrelevant probe and sections may be treated with RNAseA prior tohybridization, to assess spurious hybridization.

Thus, for instance, as discussed for uteroglobin protein, normal tissueexhibits high concentrations of uteroglobin mRNA in the prostatic,breast, or lung epithelial cells. Biopsy material from malignant tumorsshows significant decreases in the concentration of mRNA uteroglobin incells. Furthermore, aggressive malignant tumors exhibit significantlyless hybridization to a uteroglobin mRNA-specific hybridization probethan do non-aggressive malignant tumors.

Again, the differentiation between aggressive and non-aggressivemalignant cancers permits an early diagnosis, prognosis and treatment ofprostate cancer.

aberrant mRNA or DNA

Some metastatic tissues of prostatic, breast, or lung origin exhibitseemingly normal hybridization to a uteroglobin-specific probe, eventhough cells in the same tissue do not synthesize much, if any,uteroglobin protein. These cells typically exhibit aberrant uteroglobinmRNA, rather than decreased uteroglobin mRNA. For instance, aberrantsplicing has been demonstrated in at least one human prostaticcarcinoma.

Splicing and other aberrations in mRNAs of cells of metastatic tissuecan be determined by northern and southern blotting techniques and byPCR techniques. These techniques also are well known to those of skillin the art and can be applied readily to the determination of mRNA incells of biopsy material in accordance with the present invention.

Techniques employed to assess restriction fragment length polymorphisms(“RFLP”) can be applied to detect some mutations associated withaberrant splicing patterns. The assessment always can be made on themRNA, but in some dysfunctions it can be made on the genomic DNA aswell. The mRNA or DNA can be amplified prior to RFLP analysis, as well,using PCR or other suitable technique.

In addition to RFLP techniques, SSCP can be used to detect aberrantsplicing of messages, such as uteroglobin-specific mRNA. For thispurpose, a target mRNA, such as uteroglobin mRNA, is amplified byreverse transcriptase-mediated PCR. The double-stranded amplified DNA isdenatured and run on gels in which mobility is quite sensitive to smallchanges in secondary structure.

Yet another technique that can be employed to determine aberrantsplicing, among other things, is ligase mediated PCR. This techniquealso is well known to those of skill in the art, and techniques suitableto the analysis and determination of mRNAs and genomic aberrations thathave been described in the literature readily can be applied to thedetermination of aberrant mRNAs in cells of tumor.

In regard to all of the foregoing, the determination of mRNA and genomicDNA in epithelial cells in biopsy material is preferred. Particularlypreferred in this regard is prostatic, breast, or lung biopsy material.

Among probes and hybridization targets for determination of degree ofintegrin-mediated maintenance of cell invasive capacity of tumors bydetermination of target mRNA and DNA are probes specific for uteroglobinmRNA or for aberrations of uteroglobin mRNA or uteroglobin-encoding DNAindicative of altered expression of uteroglobin and, therefore, ofdegree of integrin-mediated maintenance of cell invasive capacity.

FIDUCIARIES

Fiduciaries may be developed in accordance with this aspect of theinvention, to guide interpretation of results. In this regard, a proteinor mRNA in accordance with the foregoing may be determined in biopsymaterial from representative tumors of a specific type, characteristicof a specific degree of integrin-mediated maintenance of cell invasivecapacity.

Characterization in this regard may benefit from hindsight, followingthe actual course of tumor progression in patients as they undergotreatment and thereafter. Control results characterizing a graded seriesof degree of integrin-mediated maintenance of cell invasive capacityalso may obtained from cell culture studies, as described elsewhereherein, illuminated in the examples below.

The determination of a protein or mRNA, or other agent, as set outabove, in a variety of tumors of known metastatic characteristic, andthe correlation of the determinations with degree of integrin-mediatedmaintenance of cell invasive capacity is another preferred embodiment ofthe present invention.

KITS

Reagents for carrying out the methods described above may beincorporated into kits for use in determining the degree ofintegrin-mediated maintenance of cell invasive capacity of a tumor. Allof the techniques and reagents discussed herein with regard to thedetermination of degree of integrin-mediated maintenance of cellinvasive capacity, including reagents and methods set out in theexamples below may be included in these kits. Preferred kit componentsgenerally are those that detect the agents discussed elsewhere herein,particularly as discussed in the foregoing sections pertaining todetermination of a protein or mRNA diagnostic of degree ofintegrin-mediated maintenance of cell invasive capacity.

The kits also may include one or more control results, such as referenceslides of immunocytochemical results characteristic of a tumors withhigh and low degree of integrin-mediated maintenance of cell invasivecapacity, or reference slides of in situ hybridization resultscharacteristic in the same regard. Preferably in this regard, are kitsthat include a control series for interpreting results. The control maybe in the form of one or more photographs or may be depicted in otherways, including written descriptions. In addition, the control may behighly tumor-type-specific or it may be applicable to related types oftumors.

ANTI-METASTATIC AGENTS AND METHODS

The invention disclosed herein provides agents and methods forinhibiting or preventing metastasis. The following discussionillustrates the invention in this respect.

Anti-metastatic agents

In particular, in accordance with this aspect of the invention, whichinclude compounds that restore integrin-mediated maintenance of cellinvasive capacity of a tumor of epithelial cell origin in an organismare administered by a route and in an amount effective to prevent orinhibit metastasis of the tumor.

Effectors of integrin-mediated maintenance of cell invasive capacity

Without being limited to any theory of the invention, applicants notethat uteroglobin binds to integrin and thereby modulatesintegrin-mediated maintenance of cell invasive capacity. In aggressivemalignant tumors of epithelial origin an aberrant form of uteroglobin isexpressed in place of the normal non-aberrant form of uteroglobin. Theaberrant uteroglobin is an incompetent effector of normalintegrin-mediated maintenance of cell invasive capacity. Thereby, theaberrant uteroglobin contributes to the invasion and metastasis oftumors. In some aspects of the invention, administering non-aberrantforms of uteroglobin to the tumor cells restores normalintegrin-mediated maintenance of cell invasive capacity of tumor cellsof epithelial cell origin, and thereby inhibits or extinguishes degreeof tumor invasion and metastasis.

Uteroglobins

Uteroglobin, also called blastokinin, was first discovered as a majorprotein component of the rabbit uterine fluid during early pregnancy.The human counterpart to rabbit uteroglobin was first found innonciliated Clara cells in the distal bronchiole airway and wasoriginally designated Clara cell 10 kD protein, abbreviated as “CC10.”Uteroglobin also has been detected in humans in the uterus, respiratorytract, and prostate gland, by immunohistochemical methods.

The complementary DNA for human uteroglobin (CC10) has been cloned andits sequence has been determined, as reported in Singh et al., BBA 950:329-337 (1988), incorporated by reference herein in its entirety.

Uteroglobin has been purified to homogeneity by at least two groups andit has been structurally and functionally characterized in considerabledetail. In brief, uteroglobin occurs in rabbits as a dimer of twoidentical chains. The monomers are 70 amino acids long. They arearranged antiparallel to one another in the dimer. Also, in the dimerthey are covalently linked by two symmetrical disulfide bonds, formedbetween ‘Cys-3’ and “Cys-69” and, reciprocally, between ‘Cys-69’ and“Cys-3” (where ′ designates one chain in the dimer and ″ designates theother chain). Each monomer chain contains four α-helical segments and aβ-turn, the later at Lys-26 to Gln-29. The structure, function andactivities of uteroglobin has been reviewed, for instance, by Miele etal., Endocrine Reviews 8: 474-490 (1987), which is incorporated byreference herein in its entirety.

Uteroglobin is an effector of integrin-mediated maintenance of cellinvasive capacity, as shown by in vitro assays. Generally, it has beenthought to have immunomodulatory or anti-inflammatory activities, orboth, that act to protect the wet epithelia of organs that communicatewith the external environment. Uteroglobin expression issteroid-sensitive and its secretion in the endometrium has been shown tobe stimulated by progesterone. Uteroglobin also has been reported tohave an anti-chemotactic effect on neutrophils and monocytes.Uteroglobin has not been seen as playing a role in cancer or metastasis.Thus, it was surprising to find that uteroglobin, in accordance with thepresent invention, can be used to inhibit or prevent metastasis of atumor of epithelial cell origin in an organism.

Without being bound to any theory of the mechanism by which uteroglobininhibits metastasis, it appears, as the Examples show, that theinhibitory action of uteroglobin on metastasis results from modulationof integrin-mediated maintenance of cell invasive capacity. Inaggressive malignant tumors of epithelial origin an aberrant form ofuteroglobin is expressed in place of the normal non-aberrant form ofuteroglobin. The aberrant uteroglobin is an incompetent effector ofnormal integrin-mediated maintenance of cell invasive capacity. Thereby,the aberrant uteroglobin contributes to the invasion and metastasis oftumors. In some aspects of the invention, administering non-aberrantforms of uteroglobin to the tumor cells restores normalintegrin-mediated maintenance of cell invasive capacity of tumor cellsof epithelial cell origin, and thereby inhibits or extinguishes degreeof tumor invasion and metastasis.

Any uteroglobin may be useful in the invention that inhibitsintegrin-mediated maintenance of cell invasive capacity and therebyinhibits or prevents metastasis of a tumor of epithelial cell origin.Uteroglobins for use in the invention may be recovered from naturalsources, it may be made by recombinant means, it may be produced bychemical techniques, it may be made by semi-synthetic methods or it maybe obtained by a combination of techniques. Methods for purifyinguteroglobin to homogeneity from a natural source have been described inNieto et al., Arch. Biochem. Biophys. 180:80-92 (1977), which is hereinincorporated by reference in its entirety. Other methods for thispurpose can be equally useful in this regard.

The most highly preferred uteroglobin for use in the invention at thepresent time is human uteroglobin. Preferably, human uteroglobin for usein the invention is made by expression of a cloned gene in a host cellin culture or in an animal. Techniques for expressing uteroglobin inthis way are well known to those of skill in the art.

A cDNA encoding human uteroglobin, useful toward this end, has beenisolated, sequenced and expressed in cells in culture. Methods forexpressing cloned DNAs that encode uteroglobin have been describedspecifically with regard to human uteroglobin in Mantile et al., J. BiolChem. 268: 20343-20351 (1993) and Miele et al., J. Biol. Chem. 265:6427-6435 (1990), which, as not ed below, are incorporated by referenceherein in their entirety.

Techniques for obtaining, m anipulating and expressing cloned genes toobtain uteroglobin for use in the present invention are well known tothose of skill in the art and are described in protocol-like detail in avariety of laboratory manuals. For instance, such methods are set forthin Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2ND Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989),the entirety of which is herein incorporated by reference.

Muteins and peptide analogs

Techniques such as those described in the foregoing manual can be usedto make variants and analogs of uteroglobin and other proteins useful inthe invention. Recombinant DNA methods, chemical synthetic methods,enzymatic methods and mixed methods for making, altering and utilizingmuteins and peptide analogs are well known and are described here onlybriefly to illustrate their applicability to the present invention.

-muteins

It will be appreciated by those of skill that cloned genes readily canbe manipulated to alter the amino acid sequence of a protein. The clonedgene for human uteroglobin can be manipulated by a variety of well knowntechniques for in vitro mutagenesis, among others, to produce variantsof the naturally occurring human protein, herein referred to as muteins,that may be used in accordance with the invention.

The variation in primary structure of muteins of uteroglobins useful inthe invention, for instance, may include deletions, additions andsubstitutions. The substitutions may be conservative ornon-conservative. The differences between the natural protein and themutein generally conserve desired properties, mitigate or eliminateundesired properties and add desired or new properties. In the presentinvention the muteins generally are those that maintain or increaseanti-metastatic activity. Particularly, uteroglobin muteins are aminoacid sequence variants of uteroglobin that maintain or increase theanti-metastatic activity of uteroglobin.

-peptide analogs

Similarly, techniques for making small oligopeptides and polypeptidesthat exhibit activity of larger proteins from which they are derived (inprimary sequence) are well known and have become routine in the art.Thus, peptide analogs of proteins of the invention, such as peptideanalogs of uteroglobin that exhibit anti-metastatic activity also areuseful in the invention.

Mimetics

Mimetics also can be used in accordance with the present invention toprevent or inhibit metastasis of tumors. The design of mimetics is knownto those skilled in the art, and generally are understood to be peptidesor other relatively small molecules that have an activity the same orsimilar to that of a larger molecule, often a protein, on which they aremodeled.

Thus, by way of illustration, uteroglobin mimetics, for instance, can beused in accordance with the present invention in the same manner asuteroglobin itself, to prevent or inhibit metastasis of a tumor.

The design of such mimetics can be based on the structure-functionrelationship of uteroglobin. By studying the effect of mutations onanti-metastatic activity of uteroglobin the sites in the proteinresponsible for anti-metastatic activity can be identified. In vitromutagenesis procedures that can be used to systematically alter clonedgenes, such as cDNAs encoding uteroglobin and other proteins withanti-metastatic activity of the present invention, are described inSambrook et al. (1988). Systematically mutagenized proteins, alsoreferred to as muteins as noted elsewhere herein, can be produced usingsuch altered DNA by standard methods for expression cloned genes inorganisms to produce heterologous proteins. Such methods are well knownto those of skill and are described in, for instance, Sambrook et al.(1988) referred to herein above. The muteins so produced then can beassayed for anti-metastatic activity, using in vitro or in vivo assaysthat model or measure metastatic activity. Suitable methods aredescribed herein and illustrated in the examples below.

Methods for determining aspects of protein structure also are well knownto those of skill in the art. To some extent, the structure of a givenprotein can be approximated by analogy to structures of relatedproteins. Physical and chemical information about a protein structurecan be obtained by a wide variety of well known techniques, includingactive site modification techniques, NMR, and X-ray crystallography.

This information can be combined with information from studies thatcorrelate structural alterations with changes in activity, such as themutagenesis studies described above, to generate a map of the shape andchemical functions important to a given activity of a protein.

Molecules that mimic the shape and chemical functionality that providethe desired activity, then can be designed and synthesized. Computermodeling methods that can be employed toward this end, as well asmethods of organic synthesis, peptide synthesis and for the synthesis ofother classes of compounds that can be used to produce mimetics inaccordance with this aspect of the invention are well known to those ofskill in the art.

Once a mimetic has been designed and synthesized, it can be assayed foranti-metastatic activity using techniques for this purpose, such asthose described elsewhere herein.

Results of activity studies and of structural studies of the mimeticsthemselves can be used to design further mimetics that are moreeffective, have fewer undesirable side effects, or have additionalactivities, such as by combining two mimetics in a single molecule.

In the same manner as for uteroglobin, mimetics can be designed forother compounds that have anti-metastatic activity. Preferred in thisregard, as described above, as anti-metastatic mimetic compounds thatinhibit integrin-mediated maintenance of cell invasive capacity ofcancer cells of epithelial cell origin. Particularly preferred aremimetics that are effectors of integrin-mediated maintenance of cellinvasive capacity. In this regard, mimetics of uteroglobin areespecially preferred. Among the most highly preferred mimetics in thisregard are mimetics of human uteroglobin.

Small molecule drugs

Compounds other than the proteins, muteins, protein-derived peptides,mimetics and the like discussed above, that inhibit integrin-mediatedmaintenance of cell invasive capacity of cancers of epithelial cellorigin also may be useful in the present invention. Among such compoundsare certain small organic molecules, which may be mimetics, thatspecifically bind to integrin and therby effect integrin-mediatedmaintenance of cell invasive capacity. Inhibition may be mediated byinhibition of other enzymes or intermediates involved in metabolicinteractions that result in integrin-mediated maintenance of cellinvasive capacity.

Compositions

Any non-toxic, inert and effective carrier may be used to formulatecompositions of the present invention. Well known carriers used toformulate other therapeutic compounds for administration to humansparticularly will be useful in the compositions of the presentinvention. Pharmaceutically acceptable carriers, excipients and diluentsin this regard are well known to those of skill, such as those describedin the MERCK INDEX, 11th Ed., Budavari et al., Eds., Merck & Co., Inc.,Rahway, N.J. (1989), which is incorporated by reference herein in itsentirety. Examples of such useful pharmaceutically acceptableexcipients, carriers and diluents include distilled water, physiologicalsaline, Ringer's solution, dextrose solution, Hank's solution and DMSO,which are among those preferred for use in the present invention.

In particular, for instance Mantile et al., J. Biol Chem. 268:20343-20351 (1993), incorporated by reference hereinabove, report onsterile formulated, lyophilized uteroglobin that may be useful inpreparing uteroglobin compositions of the invention.

Cancers

Methods and compositions of the present invention may be applied to thetreatment of a variety of cancers of epithelial cell origin. Among theseare metastatic cancers of breast, lung, colon, bladder, prostate,gastrointestinal track, endometrium, tracheal-bronchial tract, pancreas,liver, uterus, nasopharynges and the skin. An especially preferredtarget is prostate cancer, particularly prostate cancer of epithelialcell origin.

The following detailed discussion of prostate cancers is provided inillustration of the compositions and methods of the invention not onlyas to prostate cancers, but also other cancers that may be treated inanalogous or identical fashion, in accordance with the presentinvention.

Prostatic adenocarcinoma

Adenocarcinoma of the prostate is one of the most common malignancies.It is estimated that 240,000 new cases of prostate cancer will bediagnosed in the United States in 1995, and that it will cause more than50,000 deaths during the year. In fact, prostate adenocarcinoma is thesecond leading cause of cancer-related mortality in the United States.

With prostate cancer, as with all solid tumors, it is the metastaticencroachment of the tumor on other vital function that causes the demiseof the patient. Approximately 10% of patients are diagnosed initiallywith metastatic disease. Ultimately, 30-40% of patients with this cancerwill develop metastatic disease. Once metastasis occurs there is a thecancer follows a relentless progression.

Invasion is a prerequisite for migration of tumor cells. In connectivetissue, stroma and basement membranes form the major physical barriersto the migration process. Invasion of the local extracellular matrix(ECM) by tumor cells thus can be marked as the first step in metastasis.The sequential biochemical mechanism of ECM invasion first involves cellattachment to specific components of ECM followed by a progressivecascade of proteolytic dissolution. Prostate cancers which grow to acritical size exhibit extracapsular invasion and metastasize to specificanatomical sites apparently in response to stromal cell secretoryproteins which are necessary for their growth and proliferation.Invasive migration of tumor cells within the prostate gland may occur asa function of chemokinesis along anatomical paths of least resistance,such as the perineural duct. Further establishment of metastasis reliesupon successful penetration of the circulatory or lymphatic system,followed by vessel extravasation at the secondary organ, whichfrequently is bone tissue for cancers of prostatic origin. Nearly all ofthese steps, including attachment, matrix degradation and migration, canbe modeled experimentally in vitro by measuring invasion of areconstituted basement membrane (RBM) barrier in response tofibroblast-conditioned medium (FCM) which serves as a chemo-attractant.

In vivo, of growth and proliferation of prostate tumor cells primarilyis responsive to stromal cell (fibroblast) secretory proteins.Extracapsular invasiveness of prostate tumor cell can be modeled bymigration of tumor cells in vitro into reconstituted basement membrane(RBM) in the presence and absence of a chemoattractant, such asfibroblast conditioned medium (FCM). The assay determines cells thathave attached to the RBM, degraded the RBM enzymatically and, finally,cells that have towards the FCM side of the membrane. The events in thein vitro invasion assay comport with the important steps observed in themetastatic spread of tumor cells through the basement membrane in vivo.

Prostate tumors frequently initially metastasize to regional lymphnodes, having disseminated through the lymphatic circulation. They alsospread to other sites through the vascular system, which is extensivelyinterconnected to the lymphatics. The final site of formation ofmetastasis is a function of a number of parameters, including: (i) thefirst capillary bed encountered by blood vessels draining the tumor, and(ii) organ preference of the tumor cells with respect to characteristicsof specific tissues that nurture attachment and growth of tumor cellswith degree of integrin-mediated maintenance of cell invasive capacity.

Degree of integrin-mediated maintenance of cell invasive capacity ofprostate cancers of epithelial cells origin can be modulated bycompositions and methods of the invention. In particular, metastasis ofthese cancers can be inhibited by human uteroglobin, as shown by theexamples set out herein below.

Route of administration

Therapeutic treatment with uteroglobin can utilize any type ofadministration including topical, other non-invasive and invasive means.

Administration by non-invasive means may be by oral, intranasal ortransdermal routes, among others.

Generally, at the present time, invasive techniques are preferred.Administration by invasive techniques may be intravenous,intraperitoneal, intramuscular or directly in tumors, among others.

Administration may be by a single dose, it may be repeated at intervalsor it may be continuous. Since uteroglobin is small, easily diffusible,and relatively stable it is well suited to long-term continuousadministration, such as by perfusion pump. Where continuousadministration is applied, infusion is preferred. In this situation,pump means often will be particularly preferred for administration.Especially, subcutaneous pump means often will be preferred in thisregard.

In other situations it will be desirable to administered uteroglobin andother agents of the present invention by intramuscular self-injection ona regular basis.

Compositions and methods of the invention also may utilize controlledrelease technology. Thus, for example, uteroglobin may be incorporatedinto a hydrophobic polymer matrix for controlled release over a periodof days. Such controlled release films are well known to the art.Examples of polymers commonly employed for this purpose that may be usedin the present invention include nondegradable ethylene-vinyl acetatecopolymer and degradable lactic acid-glycolic acid copolymers. Certainhydrogels such as poly(hydroxyethylmethacrylate) or poly (vinylalcohol)also may be useful, but for shorter release cycles then the otherpolymer releases systems, such as those mentioned above.

Dose

The quantity of the active agent for effective therapy will depend upona variety of factors, including the type of cancer, means ofadministration, physiological state of the patient, other mendicantsadministered, and other factors.

Treatment dosages generally may be titrated to optimize safety andefficacy. Typically, dosage-effect relationships from in vitro studiesinitially will provide useful guidance on the proper doses for patientadministration. Studies in animal models also generally may be used forguidance regarding effective dosages for treatment of metastatic cancersin accordance with the present invention.

These considerations, as well as effective formulations andadministration procedures are well known in the art and are described instandard textbooks, such as GOODMAN AND GILMAN'S: THE PHARMACOLOGICALBASES OF THERAPEUTICS, 8th Ed., Gilman et al. Eds. Pergamon Press (1990)and REMINGTON'S PHARMACEUTICAL SCIENCES, 17th Ed., Mack Publishing Co.,Easton, Pa. (1990), both of which are incorporated by reference hereinin their entirety.

Typical therapeutic doses will be about 0.1 to 1.0 mg/kg of body weightof pure uteroglobin. The does may be adjusted to attain, initially, ablood level of about 0.1 μM.

A particular formulation of the invention uses a lyophilized form ofuteroglobin, in accordance with well known techniques. For instance, 1to 100 mg of uteroglobin may be lyophilized in individual vials,together with carrier and buffer compound, for instance, such mannitoland sodium phosphate. The uteroglobin may be reconstituted in the vialswith bacteriostatic water and then administered, as described elsewhereherein.

Administration regimen

Any effective treatment regimen can be utilized and repeated asnecessary to affect treatment.

In clinical practice, the compositions containing uteroglobin orrecombinant uteroglobin, alone or in combination with other therapeuticagents are administered in specific cycles until a response is obtained.

For patients who initially present without metastatic disease,uteroglobin-based drugs can be used as an immediate initial therapyprior to surgery and radiation therapy, and as a continuouspost-treatment therapy in patients at risk for recurrence or metastasis(based upon high marker's score, locally extensive disease, and/orpathological evidence of tumor invasion in the surgical specimen).Therapy for these patients aims, for instance, to decrease the escape ofpotentially metastatic cells from the primary tumor during surgery orradiotherapy, decrease the escape of tumor cells from undetectableresidual primary tumor, decrease tumor cell attachment to the interiorvessel wall, decrease the migration of tumor cells out of the vessel,and thereby decrease invasion into the interstitial spaces of the distalorgan.

For patients who initially present with metastatic disease,uteroglobin-based drugs can be used as a continuous supplement to, orpossible as a replacement for hormonal ablation. A goal of therapy forthese patients is to slow tumor cell escape from both the untreatedprimary tumor and from the existing metastatic lesions in order to slowthe progressive encroachment of further metastases.

In addition, the invention may be particularly efficacious duringpost-surgical recovery, where the present compositions and methods maybe particularly effective in lessening the chances of recurrence of atumor engendered by shed cells that cannot be removed by surgicalintervention.

Gene therapy

Certain embodiments of the present invention relate to anti-metastaticgene therapy. Gene therapy is a new approach to treatment of diseases.Currently, gene therapy protocols relate to therapy of certain carefullychosen disorders, including certain inherited disorders, a number ofaggressively fatal cancers and AIDS. The restricted application of genetherapy to a few disorders reflects concerns about the efficacy, safetyand ethical implications of the approach in general, and currenttechniques in particular. Despite the cautious approach mandated bythese concerns, and despite the fact that techniques for carrying outgene therapy are still in an early stage of development, results fromthe first few trials have been very encouraging, some spectacularly so.It seems certain that gene therapy techniques will improve rapidly andthat gene therapies soon will develop into an increasingly important andubiquitous modality for treating disease. (Reviewed, for instance, inTolstoshev, Ann. Rev. Pharm. Toxicol. 32: 573-596 (1993) and Morgan etal., Ann. Rev. Biochem. 62: 191-217 (1993), which are incorporated byreference herein in their entirety).

The delivery of a variety of therapeutic agents clearly will beaccomplished by gene therapy techniques. Many of the procedures now inuse or under current development for gene therapy may be used inaccordance with the present invention to prevent or inhibit metastasis.Additional techniques that will be developed in the future similarlywill be found useful in the present invention. The following discussionis illustrative of the use of gene therapy techniques to prevent orinhibit metastasis in accordance with the present invention.

By gene therapy, in the following discussion, generally is meant the useof a polynucleotide, in a cell, to achieve the production of an agentand the delivery of the agent to a tumor in situ, i.e., in a patient, toengender an anti-metastatic effect. The agent may itself be aanti-metastatic agent or it may engender the production of ananti-metastatic agent upon introduction into the patient.

Approaches to genetic therapy currently being developed, which can beused in accordance with this aspect of the invention disclosed herein,often are grouped into two major categories: ex vivo and in vivotechniques.

Ex vivo techniques generally proceed by removing cells from a patient orfrom a donor, introducing a polynucleotide into the cells, usuallyselecting and growing out, to the extent possible, cells that haveincorporated, and, most often, can express the polynucleotide, and thenintroducing the selected cells into the patient. Cells that target tumorcells in vivo, including tumor cells that have migrated from primary orsecondary tumor sites, generally are preferred in this type of genetherapy.

In addition, as described further below, the polynucleotide may beintroduced directly into the patient. The polynucleotide in this casemay be introduced systemically or by injection into a tumor site. Thepolynucleotide may be in the form of DNA or RNA, alone or in a complex,or in a vector, as discussed further below.

The polynucleotide may be in any of a variety of well-known forms, forinstance, a DNA, a DNA fragment cloned in a DNA vector, a DNA fragmentcloned in DNA vector and encapsidated in a viral capsid.

The polynucleotide may be an RNA or a DNA. More typically it is a DNA.It may include a promoter, enhancer and other cis-acting control regionsthat provide a desired level and specificity of expression in the cellsof a region operably linked thereto that encodes an RNA, such as ananti-sense RNA, or a protein. The polynucleotides may contain severalsuch operably linked control and encoding regions for expression of oneor more mRNAs or proteins, or a mixture of the two.

Preferred in this regard are polynucleotides that encode theanti-metastatic agents described herein above. As noted in the foregoingdiscussion, effectors of integrin-mediated maintenance of cell invasivecapacity are preferred. Preferred are uteroglobins, especially preferredis human uteroglobin.

Muteins and polypeptide analogs of protein effectors also are useful inthe invention and may be encoded by polynucleotides for gene therapy toinhibit metastasis. In this regard, muteins and polypeptide analogs ofthe foregoing preferred embodiments also are preferred in this aspect ofthe invention.

In addition, peptide mimetics that can be encoded by a polypeptide forsynthesis in cells can be used in accordance with this aspect of theinvention. Preferred embodiments in this regard those set out above.

The polynucleotide may be introduced into cells either ex vivo or invivo, including into the tumor in situ. A variety of techniques havebeen designed to deliver polynucleotides into cells for constitutive orinducible expression, and these routine techniques can be used in genetherapy of the present invention as well. Polynucleotides will bedelivered into cells ex vivo using cationic lipids, liposomes or viralvectors. Polynucleotides will be introduced into cells in vivo,including into cells of tumors in situ, using direct or systemicinjection. Methods for introducing polynucleotides in this manner caninvolve direct injection of a polynucleotide, which then generally willbe in a composition with a cationic lipid or other compound or compoundsthat facilitate direct uptake of DNA by cells in vivo. Such compositionsmay also comprise ingredients that modulate physiological persistence.In addition, polynucleotides can be introduced into cells in vivo inviral vectors.

Genetic therapies in accordance with the present invention may involve atransient (temporary) presence of the gene therapy polynucleotide in thepatient or the permanent introduction of a polynucleotide into thepatient. In the latter regard, gene therapy may be used to repair adysfunctional gene to prevent or inhibit metastasis.

Genetic therapies, like the direct administration of agents discussedabove, in accordance with the present invention may be used alone or inconjunction with other therapeutic modalities.

Combined with other treatments

Uteroglobin may be used in conjunction with other treatment modalities.Other common treatment modalities are discussed below specifically byreference to prostate cancer. It will be appreciated that similarconsideration will apply to treatment of other metastatic cancers. Thepresent invention may be used in conjunction with any current or futuretherapy.

Surgery and Radiation

In general, surgery and radiation therapy are employed as potentiallycurative therapies for patients under 70 years of age who present withclinically localized disease and are expected to live at least 10 years.Neither treatment modality has a significant role in the management ofmetastatic diseases, and neither treatment is generally performed ifmetastasis is present at initial diagnosis.

Approximately 70% of newly diagnosed prostate cancer patients fall intothis category. Approximately 90% of these patients (63% of totalpatients) undergo surgery, while approximately 10% of these patients (7%of total patients) undergo radiation therapy.

Histopathological examination of surgical specimens reveals thatapproximately 63% of patients undergoing surgery (40% of total patients)have locally extensive tumors or regional (lymph node) metastasis thatwas undetected at initial diagnosis. These patients are at asignificantly greater risk of recurrence or metastasis. Approximately40% of these patients will actually develop recurrence or metastasiswithin 5 years after surgery. Results after treatment with radiation areeven less encouraging. Approximately 80% of patients who have undergoneradiation as their primary therapy have disease persistence or developrecurrence or metastasis within 5 years after treatment.

Currently, surgical and radiotherapy patients generally do not receiveany immediate follow-up therapy. Rather, they typically are monitoredfor elevated Prostate Specific Antigen (“PSA”), which is the primaryindicator of recurrence or metastasis.

Thus, there is considerable opportunity to use the present invention inconjunction with surgical intervention.

Hormonal Therapy

Hormonal ablation is the most effective palliative treatment for the 10%of patients presenting with metastatic disease at initial diagnosis.Hormonal ablation by medication and/or orchiectomy is used to blockhormones that support the further growth and metastasis of prostatecancer. With time, both the primary and metastatic tumors of virtuallyall of these patients become hormone-independent and resistant totherapy. Approximately 50% of patients presenting with metastaticdisease die within 3 years after initial diagnosis, and 75% of suchpatients die within 5 years after diagnosis.

In this regard, it may be worth noting that the natural uteroglobin geneis dependent upon hormones for expression, and hormonal ablation maydecrease expression of the endogenous uteroglobin gene, both in tumorcells and in the normal tissue surrounding the tumor. Auteroglobin-deficient state could render the patient more susceptible tosuccessful metastasis. Continuous supplementation with uteroglobin-baseddrugs may be used to prevent or reverse this potentiallymetastasis-permissive state from developing in hormonal therapytreatment modalities.

Chemotherapy

Chemotherapy has been more successful with some cancers than withothers. It is likely that the combination of chemotherapy with therapiesof the present invention in some cases will be synergistic. Chemotherapycurrently has little effect on prostate cancer and is used only as alast resort, with universally dismal results.

Immunotherapy

The present invention also can be used in conjunction withimmunotherapies. Not only may the methods and compositions hereindisclosed be used with the increasing variety of immunological reagentsnow being tested or used to treat cancer, but it also may be used withthose that come into practice in the future. The present invention thusmay be used with immunotherapies based on polyclonal or monoclonalantibody-derived reagents, for instance. Monoclonal antibody-basedreagents are among those most highly preferred in this regard. Suchreagents are well known and are described in, for instance, RitterMONOCLONAL ANTIBODIES—PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS,Ritter et al., Eds., Cambridge University Press, Cambridge, UK (1995),which is incorporated by reference herein in its entirety. Radiolabelledmonoclonal antibodies for cancer therapy, in particular, also are wellknown and are described in, for instance, CANCER THERAPY WITHRADIOLABELLED ANTIBODIES, D. M. Goldenberg, Ed., CRC Press, Boca Raton,Fla. (1995), which is incorporated by reference herein in its entirety.

Cryotherapy

Cryotherapy recently has been applied to the treatment of some cancers.Methods and compositions of the present invention also can be used inconjunction with an effective therapy of this type.

Compositions comprising several active agents

According to another aspect of the invention, pharmaceuticalcompositions of matter useful for inhibiting cancer metastases areprovided that contain, in addition to the aforementioned compounds, anadditional therapeutic agent. Such agents may be chemotherapeuticagents, ablation or other therapeutic hormones, antineoplastic agents,monoclonal antibodies useful against cancers and angiogenesisinhibitors. The following discussion highlights some agents in thisrespect, which are illustrative, not limitative. A wide variety of othereffective agents also may be used.

Among hormones which may be used in combination with the presentinvention diethylstilbestrol (DES), leuprolide, flutamide, cyproteroneacetate, ketoconazole and amino glutethimide are preferred.

Among antineoplastic and anticancer agents that may be used incombination with the invention 5-fluorouracil, vinblastine sulfate,estramustine phosphate, suramin and strontium-89 are preferred.

Among the monoclonal antibodies that may be used in combination with theinvention CYT356 is preferred.

The present invention is further described by reference to thefollowing, illustrative examples.

EXAMPLE 1 Preparation of Human Uteroglobin by Gene Expression

Human uteroglobin was purified from E. coli cells expressing afull-length cDNA. The methods for obtaining the cDNA, constructing itinto a vector for expression in host, expressing the construct andpurifying the protein all involve art routine techniques. Such methodsare described specifically with regard to human uteroglobin in Singh etal., BBA 950: 329-337 (1988), Mantile et al., J. Biol Chem. 268:20343-20351 (1993) and Miele et al., J. Biol. Chem. 265: 6427-6435(1990), which are incorporated by reference herein in their entirety.

Briefly, in the present illustrative example, which followed thetechniques set out in the foregoing references, a clone containing afull-length cDNA encoding human uteroglobin in the well known vectorpGEM4Z was digested with PstI. (pGEM4Z may be obtained from Promega,Inc. Many other equally suitable vectors also are availablecommercially.) The digestion freed a 340-base pair fragment containingall of the cDNA and 53 nucleotides of the pGEM4Z polylinker. Thefragment was purified by preparative gel electrophoresis in low meltingtemperature agarose. The purified fragment was ligated into the Pstlsite of the expression vector pLD101, downstream of an induciblepromoter. The ligation and subsequent cloning produced the plasmidpGEL101. This construct was introduced into E. coli strain BL21(DE3)cells for expression of uteroglobin protein.

For expression, bacteria were cultured under routine conditions for E.coli growth, and then induced for uteroglobin expression by making themedia 0.4 5 mM in IPTG (isopropyl-1-thio-D-galactopyranoside). Afterappropriate further incubation to accumulate expressed protein, thecells were collected and then lysed. Uteroglobin was purified from thelysed cells using standard methods of size exclusion and ion exchangechromatography.

EXAMPLE 2 Cells for Assays of Degree of Integrin-mediated Maintenance ofCell Invasive Capacity

The cell lines used in the illustrative embodiments herein discussed arewell known and readily available. The four cell lines of the presentexamples all were derived from human prostate cancer and are ofepithelial cell origin. TSU-Pr1, DU-145 and PC3-M areandrogen-independent. LNCaP is androgen-sensitive.

DU-145 is described in Stone et al., Int. J. Cancer 21: 274-281 (1978),which is incorporated herein by reference in its entirety. The cell lineis available from a variety of sources including, for instance, theAmerican Type Culture Collection (Rockville, Md.).

LNCaP is described in Horoszewicz et al., Cancer Res. 43: 1809-1818(1983), which is herein incorporated by reference in its entirety. Thiscell line may be obtained from, for instance, the American Type CultureCollection (Rockville, Md.).

PC3-M is described in Kaighn et al., Invent. Urol. 11:16-23 (1976) whichis herein incorporated by reference in its entirety.

TSU-Pr1 is described in Hzumi et al., J. Urology 137:1304-1306 (1987)which is incorporated by reference in its entirety.

Cells of each line were grown and maintained in monolayer culture inαMEM (minimal essential medium) supplemented with glutamine, 10% fetalbovine serum, penicillin (100 units/ml) and streptomycin (100 (g/ml).Cultures were incubated at 37° C. in 5% CO₂/95% air. Media was replacedevery second day.

EXAMPLE 3 General Assay for Invasiveness of Cells

1. Culture

As described briefly below, invasiveness of cells was assayed by themethods described in Albini et al., Cancer Research 47: 3239-3245(1987), which is incorporated herein by reference in its entirety.Invasiveness assays and other methods for assessing anti-metastaticaffects, as discussed herein below are described in Leyton et al.,Cancer Research 54:3696-3699 (1994) which is incorporated by referenceherein in its entirety.

Cells in logarithmic phase were detached from the growth surface bybrief exposure to 0.25% trypsin, 0.25% EDTA, collected and centrifugedat 800×g for 5 min. The pellet was resuspended in SF medium, counted andseeded into 6 mm dishes, 1.5×10⁶ cells per dish. The cells then wereincubated for 24 hr in media containing zero, 0.01, 0.1 and 1.0 μMuteroglobin. After the incubation cells were gently collected using arubber policeman and assayed for invasiveness.

Fibroblast conditioned media (FCM) served as a chemo-attractant tostimulate invasion. It was prepared by culturing proliferating 3T3 cellsfor 24 hr. in SF medium and then collecting the media, free of cells.The cell free media thus obtained served as FCM.

Invasiveness was measured using a polycarbonate membrane precoated witha reconstituted basement membrane. Well known RBMs are suitable for thispurpose. For example, Albini et al., supra describes RBM of the typeemployed for these experiments.

Assays were performed in blind-well Boyden chambers. The lowercompartment of each chamber was filled with 220 μl of FCM, aschemo-attractant, or 220 μl serum-free media, as control for basalinvasiveness. A polycarbonate membrane (12-μm pore size), coated with 25μg/50 μl RBM, was placed over the lower compartment. Tumor cells forassay were added to the upper compartment, 3.0×10⁵ cells per well, andthe chambers then were incubated at 37° C. for 6 hr.

(Reconstituted basement membrane preparations for use in accordance withthe foregoing assay are readily available from numerous commercialsuppliers. One suitable example membrane in this regard is “MATRIGEL”sold by Collaborative Biomedical Products of Bedford, Mass.)

2. Quantitation

Invasive activity was measured by the number of cells that penetratedthe RBM, as determined by a technique involving crystal violet stainingdeveloped for use with the Boyden chamber. The technique, summarizedbelow, is well known and is described, for instance, in Frandson et al.,Fibrinolysis 6(Supp4): 71-76 (1992), which is incorporated by referenceherein in its entirety.

The RBM-coated membrane was removed from each chamber at the end of theincubation period. The filters were pinned down to a wax plate, keepingthe surface with the invading cells upward. The cells were stained onthe filters with 0.5% crystal violet in 25% methanol for 10 minutes.Then, the filters were rinsed in distilled water, four times or untilcrystal violet no longer leached into the wash water. After the wash,the surface of each filter that had been in contact with the wax platewas carefully wiped clean with a moist cotton swab, to removenonmigrating cells. The filters then were placed in a 24-well clusterplate and dried overnight.

Crystal violet in the invading cells on each filter was extracted twicefor 10 minutes into 500 μl aliquots of 0.1 M sodium citrate, 50%ethanol. The amount of crystal violet in the extract was analyzed byabsorbance at 585 nm, using a standard spectrometer.

3. Analyses

Assays were carried out in triplicate for each data point.

Variance between control and test groups was analyzed for significanceusing the standard repeated measures test for analysis of variance. P<0.01 generally was considered indicative of a significant effect, withexceptions, as noted elsewhere herein.

EXAMPLE 4 Correlation of Optical Density with Cell Counts, and Assay ofBasal Invasiveness

To calibrate optical density of the crystal violet extracts against thenumber of migrating cells on filters, cells were counted, seeded atknown densities on filters, incubated to allow attachment and washed.One set of a duplicate set of plates was used for cell counting. Theother set was used for staining. For counting, cells were released fromthe filters by mild trypsinization and then counted using an automatedcell counter. For staining, after washing, the cells were stained andcrystal violet stain then was extracted from the stained cells, asdescribed in EXAMPLE 3. The optical densities of the extracts weremeasured by standard spectroscopy. The optical density determined foreach filter extract was matched with the number of cells attached to itscompanion filter, as determined by direct counting. These paired datapoints served to correlate optical density with cell counts. plottedthat displayed the

In accordance with the foregoing procedure, several densities of DU-145cells were seeded into the top chamber of the Boyden jars. The jars wereset up in pairs, and for each pair dye uptake by the cells was measuredon one filter and the number of cells was counted on the other filter,as described above.

The results are shown in Table I, which sets out the number of cellsmigrating to the lower face of the filters as a function of the numberof cells seeded in the upper chamber. The number of cells migrating tothe lower filter surface also is set out as a percentage of the totalnumber of cells seeded in the top chamber. The data in the Table are themeans of triplicate determinations for each condition, and the indicatedvariance is the standard error of the mean.

At cell seedings greater than 2×10⁵ approximately 22% of cells invadedthe RBM and migrated through the filter in 6 hr. Seeding higher numbersof cells did not increase invasiveness at 6 hr. (Not shown.)

The cells were counted using an automated cell counter, such as the“COULTER MULTISIZER” made by Coulter, Inc. of Hialeah, Fla. By theseexperiments it was determined that an absorbance of 0.1 units of thecrystal violet extract corresponds to approximately 5000 cells thatmigrated through the filter.

Similar procedures can be applied to calibrate the migration assay forother cells, to employ the assay to measure the therapeutic activity ofother compositions of the present invention.

TABLE 1 Relationship between cell invasion and optical absorbance CellsCells seeded O.D. units^(a) invading Percentage (× 10³) (585 nm) (× 10¹)invasion 100 0.60 ± 0.1 31.8 ± 0.2 31.8 ± 0.2 200 1.50 ± 0.2 42.0 ± 2.121.2 ± 1.0 300 1.20 ± 0.2 72.3 ± 2.7 23.0 ± 0.4 ^(a)O.D. unitcorresponds to approximately 5,000 cells.

As shown in FIG. 1, cells of the TSU-Pr1 cell line exhibited the highestrate of basal invasiveness and the highest FCM-stimulated invasiveness.The basal rate for DU-145 cells was 2-fold lower than the rate forTSU-Pr1 cells, but the rate of stimulated invasiveness was comparablefor the two cell lines. As shown in FIG. 2, basal and stimulatedinvasiveness of PC3-M cells both were 2-fold lower than that observedfor DU-145 cells. (N.B. The scale change in FIG. 2.) LNCaP cellsexhibited the lowest basal and the lowest stimulated invasiveness. FCMstimulation increased the invasiveness of this cell line 2-3-fold, alsoas shown in FIG. 2.

EXAMPLE 5 Uteroglobin Inhibits Invasiveness of Tumor Cells

The ability of uteroglobin to inhibit tumor cell invasiveness isillustrated by its effect on cell lines treated with 0.01, 0.1 or 1.0 μMuteroglobin. Each of the four cells lines was incubated for 24 hr. withthese concentrations of uteroglobin. After the incubation period thecells were rinsed and then assayed for invasiveness. Inhibition ofstimulated invasiveness then was quantitatively determined bysubtracting the basal invasiveness from FCM-stimulated invasiveness, foreach treatment group. Finally, the results were calculated as apercentage of the invasiveness of untreated control cells.

All four cell lines showed a dose-dependent inhibition of FCM-stimulatedinvasion, as shown in FIGS. 1 and 2, by the bars for FCM/UG. Notably,uteroglobin did not affect basal invasiveness, indicated by the barslabelled SFM/UG. Table 2 shows the average inhibition observed in threeindependent experiments, each of which was performed in triplicate. Theinhibition of invasiveness by uteroglobin was found to be significant atthe P<0.01 level for all conditions, except for PC3-M and TSU-Pr1 cellstreated with 0.01 μM uteroglobin, which were significant at the P<0.05level. As shown in Table 2, 1.0 μM uteroglobin inhibited invasiveness ofDU-145 cells by 60%, PC3-M cells by 88%, LNCaP cells by 92% and theTSU-Pr1 cells by 59%.

TABLE 2 Inhibition of tumor cell invasiveness by uteroglobin %Inhibition of invasion by uteroglobin Cell lines 0.01 μM 0.1 μm 1.0 μMDU-145 45.4 ± 6^(a)  49.9 ± 5^(a)  60.2 ± 11^(a) PC3-M 43.4 ± 15^(b)82.4 ± 14^(a) 87.9 ± 11^(a) TSU-Prl 33.5 ± 10^(b) 44.4 ± 11^(a) 58.9 ±8^(a)  LNCaP 71.5 ± 10^(a) 81.3 ± 6^(a)  92.3 ± 7^(a) 

EXAMPLE 6 Time Course of Inhibition of Invasiveness by Uteroglobin

The time course for suppression of invasiveness of DU-145 cells byuteroglobin was determined over a 24 hr. period. Cells were treated, asdescribed above, for 3, 6, 12, or 24 hr. and then assayed forinvasiveness. The maximum inhibition of invasiveness observed in thecells cultured in the presence of uteroglobin was 74%, at 12 hr. 50%maximum inhibition was observed after 3 hr., and at 24 hr. inhibitionwas 79% of the maximum. FIG. 4 shows these results in graphical form.

EXAMPLE 7 Uteroglobin Does Not Affect Simple Motility

Experiments carried out to assess motility per se show that uteroglobindoes not affect normal cell motility, i.e., migration in the absence ofRBM. The results show that uteroglobin specifically inhibits theinvasion-associated motility of epithelial tumor cells.Invasion-associated motility, which is implicated more specifically inmetastasis than motility per se, involves the synthesis, recruitment, oractivation of several different classes of proteolytic enzymes includingcollagenases, cathepsins, plasminogen activators and a variety ofmetalloproteinases required for degradation of basement membranes andthe ECM. The observation that uteroglobin does not alter cell motility,but inhibits FCM-stimulated invasiveness, indicates that uteroglobinspecifically can inhibit metastatic invasiveness without directlyaltering motility of normal cells. This is an advantageous property forpharmacological intervention where non-specific effects of uteroglobinon normal motility could be disadvantageous.

EXAMPLE 8 Loss of Uteroglobin Expression is an Indicator of EpithelialCell Cancer Progression

Immunohistochemical analysis of fresh frozen prostate tissues fromsurgical specimens were taken from 50 patients.

Eight slides per patient were analyzed for uteroglobin staining. Slidesfrom 26 patients showed evidence of prostate cancer, while slides fromthe remaining 24 patients showed only benign glands. The results ofTable 3 and Table 4 demonstrate uteroglobin immunoreactivity in normalprostate, benign prostatic hyperplasia (BPH), and prostatic atrophy; lowbut clearly positive expression in prostatic intraepithelial neoplasia(PIN); positive expression in cancerous glands of Gleason's pattern lessthan or equal to 2 and complete loss of uteroglobin immunoreactivity incancerous glands 3 or greater. In addition, in the one case ofmetastatic prostate cancer that we examined, the prostate cancer cellswithin the lymph node lacked uteroglobin expression.

Further, Western analysis was performed on prostate tissue from six ofthe patients in this study presenting with cancers of Gleason's grade 8or 9. The UG protein runs at around 10 kDa on an SDS polyacrylamide gel.This protein was present in normal tissue lysates but absent in thelysates taken from cancerous tissue.

TABLE 3 SUMMARY OF PATIENT DATA # OF Characteristic Detail PATIENTS N =50 AGE Range 35-76 yrs Median 60.8 yrs RACE African American 22Causcasian 28 FINAL Benign Prostatic Hypersplasia 4 PATHOLOGICAL BladderCarcinoma 2 DIAGNOSIS PIN 5 (The two Prostate CA Gleason's Grade 5 4patients with Prostate CA Gleason's Grade 6 12 bladder Prostate CAGleason's Grade 7 20 carcinoma had Prostate CA Gleason's Grade 8 2 afinal Prostate CA Gleason's Grade 9 7 diagnosis of Prostate CA Gleason'sGrade 10 1 BPH. PIN was associated with PC in all five cases; thus N =PC + BPH = 50) SLIDE Benign Prostatic Hyperplasia 42 DIAGNOSIS ProstaticAtrophy 3 (Patterns in PIN 2 glands and PC Gleason's Pattern ≧3 20 cellsin the PC Gleason's Pattern ≦2 6 field of view Fibromuscular Tissue only2 at the time of slide analysis. The numbers here represent n [patients]out of a total of 50 in which these pathologies were observed)

Table 3. Summary of Patient Data—Summary of patient characteristicsaccording to age, race, and final pathological diagnosis as determinedby surgical pathological examination of the resected organ. Alsoincluded in this table is a summary of the differing pathologies seenamong the various fields of view in different patients at the time ofslide analysis.

TABLE 4 SUMMARY OF STAINING FOR UG EXPRESSION Negative Low ModerateStrong (0) (+) (++) (+++) BENIGN 19 25 1 PIN 2 CANCER 5 1 (≦2) CANCER 20(≧3)

Table 4. Summary of staining for UG expression. Staining of the 50patients samples for UG can be summarized as follows. Of 50 samplessamples, 2 contained fibromuscular tissue, which does not express UG,and so were excluded from this table. Two samples contained cancer(Gleason's pattern ≧3), but no benign tissue, and one more samplecontained cancer (Gleason's pattern ≧3), and PIN, but no benign tissue.Thus, the remaining 23 cancers (17 Gleason's pattern ≧3, and 6 Gleason'spattern ≦2) form a subset of 45 samples that contain benign tissue.Cancers were divided into those of Gleason's pattern ≦2 which showpositivity for UG and those of Gleason's pattern ≧3, which lack stainingfor UG. Immunoreactivity is ranked as in the previous table, whereabsence of staining is marked as negative, or 0, low positivity is +,moderate positivity is ++, and strong positivity is +++.

EXAMPLE 9 Specificity of Uteroglobin Anti-metastatic Effects

The specificity of uteroglobin activity was demonstrated in DU-145cells, using myoglobin, albumin and heat inactivated uteroglobin. Cellswere treated for 24 hr with either myoglobin, albumin or uteroglobinthat had been inactivated by incubation at 55° C. for 45 min. Theresults, presented in the graph in FIG. 3, show that myoglobin, albuminand heat-inactivated uteroglobin do not effect invasive activity oftumor cells.

EXAMPLE 10 Uteroglobin Binds to Integrin in Normal Tissue

Slices of tissue from normal prostate and tumorous prostate glands werehomogenized and processed for immunoprecipitation (FIG. 5). Thehomogenate was reacted with monoclonal antibody to integrin (alpha Vsubunit) and the immunoconjugates were isolated and analyzed by SDSpolyacrylamide gel electrophoresis. After electrophoresis, the proteinswere transferred to polyvinylidine difluoride membranes byelectrotransfer and probed with antibody to human uteroglobin. Apositive uteroglobin signal was obtained in samples of normal tissuewhich was present but markedly diminished in tumor tissue (FIG. 5). Thefact that uteroglobin co-immunoprecipitated with anti-integrin antibodyindicated that uteroglobin was bound to integrin in the tissue in vivo.

EXAMPLE 11

A patient presents with metastatic adenocarcinoma of the prostate. Theadenocarcinoma appears not to have metastasized. The adenocarcinoma isremoved by surgery. Uteroglobin is administered before and after surgeryat a dose rate that reaches and then maintains a blood concentration ofuteroglobin of approximately 1 μM. After post-operative recovery, thepatient is maintained at a decreased level of uteroglobin by a regimenof periodic i.m. self-administration. No further occurrences of theadenocarcinoma develop.

EXAMPLE 12

A patient presents with metastatic adenocarcinoma of the prostate. Theadenocarcinoma appears not to have metastasized. The adenocarcinoma isremoved by surgery. Uteroglobin is administered before and aftersurgery, at a dose rate that reaches and then maintains a bloodconcentration of uteroglobin of approximately 1 AM. After post-operativerecovery, the patient is maintained at a decreased level of uteroglobinby intermittent or continuous administration by subdural pump. Nofurther occurrences of the adenocarcinoma develop.

EXAMPLE 13

A patient presents with metastatic adenocarcinoma of the prostate. Theadenocarcinoma appears not to have metastasized. The adenocarcinoma isremoved by surgery. Uteroglobin is administered before and aftersurgery, at a dose rate that reaches and then maintains a bloodconcentration of uteroglobin of approximately 1 μM. After post-operativerecovery, the patient is maintained at a decreased level of uteroglobinby intermittent or continuous administration using a transdermal patch.No further occurrences of the adenocarcinoma develop.

EXAMPLE 14

A patient presents with metastatic adenocarcinoma of the prostate. Theadenocarcinoma appears to have metastasized, but surgery still isindicated as an effective treatment modality. Tumor tissue is removed bysurgery. Uteroglobin is administered from the time, approximately, ofthe initial diagnosis and continues after surgery, i.m. and i.v., at adose rate that reaches and then maintains a blood concentration ofuteroglobin above 1 μM. After post-operative recovery, the patient ismaintained at this level of uteroglobin by a regimen of periodic i.m.self-administration. The patient is monitored carefully for intolerableadverse side-effects of high-dose uteroglobin administration. No furthertumors develop.

EXAMPLE 15

A patient presents with metastatic adenocarcinoma of the prostate. Theadenocarcinoma appears to have metastasized, but surgery still isindicated as an effective treatment modality. Tumor tissue is removed bysurgery. Uteroglobin is administered from the time, approximately, ofthe initial diagnosis and continues after surgery, i.m. and i.v., at adose rate that reaches and then maintains a blood concentration ofuteroglobin above 1 μM. After post-operative recovery, the patient ismaintained at this level of uteroglobin by a regimen of periodic i.m.self-administration. The patient is monitored carefully for intolerableadverse side-effects of high-dose uteroglobin administration. Althoughsome of the original, small tumorous masses are detected after surgery,they do not grow in size.

EXAMPLE 16

A patient presents with metastatic adenocarcinoma of the prostate. Theadenocarcinoma appears to have metastasized, but surgery still isindicated as an effective treatment modality. Tumor tissue is removed bysurgery. Uteroglobin is administered from the time, approximately, ofthe initial diagnosis and continues after surgery, i.m. and i.v., at adose rate that reaches and then maintains a blood concentration ofuteroglobin above 1 μM. After post-operative recovery, the patient ismaintained at this level of uteroglobin by a regimen of periodic i.m.self-administration. The patient is monitored carefully for intolerableadverse side-effects of high-dose uteroglobin administration. Tumorousmasses are detected after surgery, but their growth is slowed.

EXAMPLE 17

A patient presents with a tumor of the breast, of epithelial cellorigin. The tumor, which appears to be of a metastatic type, appears notto have metastasized. The tumor is removed by surgery. Uteroglobin isadministered after surgery, i.m. and i.v., at a dose rate that reachesand then maintains a blood concentration of uteroglobin of approximately1 μM. After post-operative recovery, the patient is maintained at adecreased level of uteroglobin by a regimen of periodic i.m.self-administration. No further occurrences of the tumor develop.

EXAMPLE 18

A patient presents with a breast tumor of epithelial cell origin. Thebreast tumor has metastasized. Numerous secondary tumors are detected.Insofar as possible, tumor tissue is removed by surgery. Surgicalintervention is aggressive. Uteroglobin is administered from the time,approximately, of the initial diagnosis and continues after surgery,i.m. and i.v., at a dose rate that reaches and then maintains a bloodconcentration of uteroglobin above 1 μM. After post-operative recovery,the patient is maintained at this level of uteroglobin by a regimen ofperiodic i.m. self-administration. The patient is monitored carefullyfor intolerable adverse side-effects of high-dose uteroglobinadministration. No further tumors develop in the remaining breast orelsewhere in the body.

EXAMPLE 19 Uteroglobin is not Expressed or Secreted by Lung CarcinomaCells

A 76 year old patient would undergo surgery for radical perinealprostatectomy because of a biopsy-based diagnosis of epithelialcarcinoma of the prostate with a Gleason's score of 4. Postsurgicaldefinitive pathologic diagnosis showed moderately to poorlydifferentiated adenocarcinoma, Gleason's score of 8, nodularhyperplasia, and multifocal high grade prostatic intraepithelialneoplasia (PIN). Perineural and lymphatic invasion was noted, but theseminal vesicles were free of tumor.

With informed consent and in accordance with approved procedures,prostate tissue was obtained from the diseased biopsy after removal, forevaluation of uteroglobin expression. Tissue fragments were frozen inliquid nitrogen. Individual slices of frozen tissue were sectioned bycryostat and mounted on silanated microscope slides.

Samples were fixed with 4% formalin for 3 minutes at room temperatureand washed for 10 min in phosphate buffered saline (PBS) pH 8.0.Nonspecific reactivity was blocked by incubating samples with rabbitserum (1:100 dilution) for 30 min. The samples were then exposed to a1:1,000 dilution of goat anti-human uteroglobin primary antibodyovernight at 4 degrees C. Control samples were exposed to a 1:100dilution of goat serum.

All samples were then exposed to biotinylated rabbit anti-goat antibody(1:10,000) for 30 min and then washed with PBS for 10 min. Streptavidincomplex was added for 15 min and the samples washed again with PBS for10 min. DAB was reacted with the samples for 2 min followed by standardstaining of the tissue with hematoxylin-eosin.

Slides were analyzed by two certified pathologists, who were notinformed of the identity of any slides and who carried out theiranalyses independently.

In each case, the normal prostate tissue stained strongly positive foruteroglobin in epithelial cells, especially at the intracellular luminalsurface. Stromal cells were negative. Areas of hyperplasia diagnosedindependently by both pathologists as neoplasia stained positively. Incontrast, tumorous epithelial cells exhibited little or no staining foruteroglobin, demonstrating that the tumor cells have lost the ability tosynthesize and secrete this protein.

EXAMPLE 20 Uteroglobin mRNA is not Detected or is Aberrantly Processedin Cells Derived from Metastases of Human Prostatic Tumors

RNA was isolated from normal prostate tissue and from the DU-145, PC-3and TSU-PR1 cell lines derived from metastatic human prostate tumors.The RNA was subjected to Northern blotting according to routine andstandard procedures, using a radiolabelled uteroglobin cDNA probe.Autoradiographic analysis showed that the normal tissue expresses anabundant normally processed 600 base pair mRNA uteroglobin transcript.In contrast the metastatic tumor cells either did not detectably expressthe uteroglobin transcript (DU-145 and PC-3) or expressed a grosslyaberrant transcript (TSU-PR1).

EXAMPLE 21

A patient presents with urinary obstruction and after digital rectalexam a biopsy of the prostate is taken. The initial presurgical reportassigns a Gleason's score of 3 suggesting a low-grade localized tumor. Aportion of the biopsy sample is analyzed immunohistochemically foruteroglobin expression which is found to be present in the normal tissuebut absent in the tumor cells. The diagnosis, prognosis, and plan fortherapy is appropriately altered to reflect the high probability, basedon lack of uteroglobin expression, that the tumor is actually of highergrade than initially diagnosed and probably invasive and metastatic.Uteroglobin therapy is immediately begun.

EXAMPLE 22

After radical prostatectomy, a patient presents with high grademetastatic prostatic adenocarcinoma that has become refractory tohormonal therapy. The patient refuses chemotherapy based on its dismalefficacy against prostate cancer and its devastating side effects. Insitu hybridization analysis of a tumor biopsy reveals that theuteroglobin gene is not being expressed in the tumor cells. Furtheranalysis of uteroglobin gene structure by SSCP and RFLP indicate thethat the gene is mutated and dysfunctional. The patient chooses tobecome a candidate for gene therapy. The patient is injected with anadenovirus-based plasmid expression vector containing the uteroglobingene linked to the promotor of the PSA gene which is specificallyexpressed in prostate cells. The vector is encapsulated in liposomeswhich have anti-PSA antibody fixed on the surface. The antibody-liposomecomplex binds specifically to cells secreting PSA which presumably areonly the metastatic tumor cells. The liposomes are ingested by the cellsand release the plasmids which incorporate into the cells' genomic DNAand begin expressing uteroglobin. The transfected cells expressinguteroglobin reverse their invasive phenotype, thereby ceasing furthermetastasis and are gradually destroyed by the bodies natural defenses.The metastatic tumors regress and the patient's life is prolonged.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and all suchmodification are intended to be included within the scope of thefollowing claims.

What is claimed is:
 1. A method for inhibiting metastasis of a cancer ofepithelial cell origin, comprising the step of administering uteroglobinto an organism suffering from a cancer of epithelial cell origin by aroute and in an amount effective to inhibit metastasis of said tumor. 2.A method according to claim 1, wherein said cancer of epithelial originis chosen from the group consisting of: prostate cancer, breast cancer,and lung cancer.
 3. A method according to claim 1, wherein said compoundis human uteroglobin.
 4. A method according to claim 1, wherein saidorganism is a human patient.
 5. A method according to claim 3, whereinsaid organism is a human patient.
 6. A method according to claim 1,wherein said method is used in conjunction with another treatment.
 7. Amethod according to claim 6, wherein said treatment is surgicalintervention, radiation therapy, hormonal therapy, immunotherapy,chemotherapy, cryotherapy or gene therapy.